WO2001030832A1 - Nouveau polypeptide, proteine a doigt de zinc hkznf-23, et un polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine a doigt de zinc hkznf-23, et un polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001030832A1
WO2001030832A1 PCT/CN2000/000326 CN0000326W WO0130832A1 WO 2001030832 A1 WO2001030832 A1 WO 2001030832A1 CN 0000326 W CN0000326 W CN 0000326W WO 0130832 A1 WO0130832 A1 WO 0130832A1
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Prior art keywords
polypeptide
polynucleotide
hkznf
zinc finger
finger protein
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PCT/CN2000/000326
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Bio Road Gene Development Ltd.
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Priority to AU77691/00A priority Critical patent/AU7769100A/en
Publication of WO2001030832A1 publication Critical patent/WO2001030832A1/fr

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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
    • 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 finger protein HKZNF-23, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • the zinc finger functional domain is an important characteristic structure of zinc finger protein. Its main role is to combine with the regulatory sequences of certain genes to play the role of transcriptional regulation.
  • the zinc finger domain is characterized by zinc ions coordinating the protein's folding and DNA binding characteristics.
  • the zinc finger domain is composed of 25-30 amino acids. It is generally believed that this structure can bind to 5 nucleotides. Different positions of cystine and histidine determine different types of zinc finger proteins.
  • the structure of C2H2 type zinc fingers is that the first pair of amino acids coordinated by zinc ions are cysteine residues, and the second pair of amino acids is histidine residues. Numerous experiments confirm the DM-binding properties of this zinc finger-dependent domain.
  • C2H2 zinc finger proteins are structurally characterized by having multiple zinc finger functional domains at their carboxyl ends, and exercise DNA binding through these functional domains; there can be many different types of other functional domains at the amino terminus to exercise their multiple transcriptions Regulation.
  • C2H2 zinc finger proteins are found in many species, such as yeast, Drosophila, amphibians, mammals, etc .; they also have a wide range of functions, some promote transcription, some inhibit transcription, and some are related to nerve development and embryo formation ,spermatogenesis, hematopoietic stem cell formation and differentiation, and some are related to tumor growth, tumor growth inhibition, tumor resistance and so on.
  • C2H2 Kuppe-like zinc finger protein gene from the fetal brain cDNA library and named the gene HKZNF.
  • This gene contains multiple C2H2 zinc finger functional domains, which conform to the structural characteristics of zinc finger proteins. The function of this functional domain is to bind to the regulatory sequence of the gene and perform transcriptional regulatory functions.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide. It is another object of the present invention to provide a recombinant vector containing a polynucleotide encoding a zinc finger protein HKZNF-2 3.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a zinc finger protein HKZNF-2 3.
  • Another object of the present invention is to provide a method for producing zinc finger protein HKZNF-23.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, the finger protein HKZNF-2 3.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, the terminology protein HKZNF-23.
  • Another object of the present invention is to provide a method for diagnosing and treating a disease associated with abnormality of zinc finger protein HKZNF-23.
  • a novel isolated zinc finger protein HKZNF-23 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 active fragment, or its active derivative, analog.
  • 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 70 nucleotides with a nucleotide sequence selected from the group % Identity: (a) a polynucleotide encoding the aforementioned zinc finger protein HKZNF-2 3; (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 2095 to 2736 in SEQ ID NO: 1; and (b) a sequence having 1 to 3351 in SEQ ID NO: 1 Sequence of bits.
  • FIG. 1 is a comparison diagram of amino acid sequence homology between the zinc finger protein HKZNF-2 3 of the present invention and a human C2H2 type zinc finger protein.
  • the upper sequence is the zinc finger protein HKZNF-2 3 and the lower sequence is a human C2H2 zinc finger protein.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 1 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated zinc finger protein HKZNF-2 3.
  • 2 3. 4kDa 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 HKZNF-2 3 refers to zinc finger protein HKZNF-23 which is essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify the zinc finger protein HKZNF- 2 3 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the zinc finger protein HKZNF-2 3 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, zinc finger protein HKZNF-23, 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 may be naturally purified products or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of the zinc finger protein HKZNF-23.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the zinc finger protein HKZNF-23 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by the genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III) such One in which the mature polypeptide is associated with another compound (such as A compound that prolongs the half-life of a polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence (such as a leader sequence or a secretion sequence or a polypeptide used to purify the polypeptide) formed by fusing additional amino acid sequences into a mature polypeptide Sequences or protease sequences)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes 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 with a total length of 3351 bases, and its open reading frame (2095-2736) encodes 21 3 amino acids.
  • this polypeptide is highly homologous to the zinc finger domains of other C2H2 zinc finger proteins, and that the polypeptide has the typical characteristics of the HKZNF gene family and multiple zinc finger domains.
  • Human HKZNF is a member of the Kruppe l-like (C2H2) zinc finger protein family.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding the 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 comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the present invention also relates to a polynucleotide that hybridizes to a sequence described above 50% less, preferably 70% identity).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the 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 denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the identity between the two sequences is at least 95% Above, it is more preferable that the hybridization occurs at 97% or more.
  • 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 the zinc finger protein HKZNF-23.
  • 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 HKZNF-23 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 DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • mRNA extraction There are many mature techniques for mRNA extraction, and 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 can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybrids; (2) the presence or absence of marker gene functions; (3) measuring the level of the transcript of zinc finger protein HKZNF-23; (4) ) Through immunological techniques or determination of 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 DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of the zinc finger protein HKZNF-23 gene 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 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 the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the zinc finger protein HKZNF-23 coding sequence, and a recombinant technology to produce the polypeptide of the present invention. method.
  • the polynucleotide sequence encoding the zinc finger protein HKZNF-23 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chera.
  • any plasmid and vector can be used to construct recombinant expression vectors.
  • 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 an expression vector containing a DNA sequence encoding the zinc finger protein HKZNF-23 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, 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. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a zinc finger protein HKZNF-23 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.
  • 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 insect 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 that take up DNA can be harvested after the exponential growth phase.
  • the treatment is performed using the ( 12 method, the steps used are well known in the art.
  • MgC l 2 can be used.
  • transformation can also be electroporated
  • 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 the recombinant zinc finger protein HKZNF-2 3 (Sc ience, 1 984; 224: 14 31). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and immune diseases.
  • the present invention also provides screening compounds to identify zinc finger proteins that increase (agonist) or repress (antagonist)
  • HKZNF-23 Method of Pharmacy. Agonists enhance the biological functions of zinc finger protein HKZNF-23 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing the zinc finger protein HKZNF-23 can be cultured with the labeled zinc finger protein HKZNF-23 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of zinc finger protein HKZNF-2 3 include antibodies, compounds, receptor deletions, and Analogs and so on.
  • An antagonist of the zinc finger protein HKZNF-23 can bind to the zinc finger protein HKZNF-23 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 a biological function.
  • zinc finger protein HKZNF-23 can be added to the bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between zinc finger protein HKZNF-23 and its receptor. .
  • Receptor deletions and analogs that function as antagonists can be screened in the same manner as described above for screening compounds.
  • Peptide molecules capable of binding to zinc finger protein HKZNF-23 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the zinc finger protein HKZNF-23 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 the zinc finger protein HKZNF-23 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting zinc finger protein HKZNF-23 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 Freund's adjuvant. Wait.
  • Techniques for preparing monoclonal antibodies to zinc finger protein HKZNF-23 include but are not limited to hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV- Hybridoma technology, etc.
  • Chimeric antibodies combining human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). 0
  • Existing techniques for producing single-chain antibodies can also be used to produce single chain antibodies against zinc finger protein HKZNF-23.
  • Anti-Zinc Finger Protein HKZNF-23 antibody can be used in immunohistochemistry to detect zinc finger protein HKZNF-23 in biopsy specimens.
  • Monoclonal antibodies that bind to zinc finger protein HKZNF-23 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.
  • Such as zinc finger protein HKZNF-23 high affinity monoclonal antibodies can interact with bacterial or phytotoxins (such as diphtheria toxin, ricin, red beans) Base, 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 zinc finger protein HKZNF-23 positive cells .
  • the antibodies in the present invention can be used to treat or prevent diseases related to the zinc finger protein HKZNF-23.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of zinc finger protein HKZNF-23.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of zinc finger protein HKZNF-23.
  • tests are well known in the art and include F I SH assays and radioimmunoassays.
  • the level of zinc finger protein HKZNF-2 3 detected in the test can be used to explain the importance of zinc finger protein HKZNF-23 in various diseases and to diagnose diseases in which zinc finger protein HKZNF-23 plays a role.
  • 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.
  • the polynucleotide encoding the zinc finger protein HKZNF-23 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 HKZNF-23.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express the variant zinc finger protein HKZNF-23 to inhibit endogenous zinc finger protein HKZNF-23 activity.
  • a variant zinc finger protein HKZNF-23 can be a shortened zinc finger protein HKZNF-2 3 lacking a signaling domain, although it can bind to downstream substrates, but lacks signaling activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of zinc finger protein HKZNF-23.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, and parvovirus can be used to transfer the polynucleotide encoding the zinc finger protein HKZNF-23 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a zinc finger protein HKZNF-2 3 can be found in existing literature (Sambrook, et al.).
  • a polynucleotide encoding the zinc finger protein HKZNF-23 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 the zinc finger protein HKZNF-23 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. For example, the technique of solid phase phosphoramidite chemical synthesis to synthesize oligonucleotides 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 DM sequence has been integrated downstream of the RM 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 the zinc finger protein HKZNF-2 3 can be used for the diagnosis of diseases related to the zinc finger protein HKZNF-23.
  • the polynucleotide encoding the zinc finger protein HKZNF-23 can be used to detect the expression of the zinc finger protein HKZNF-2 3 or the abnormal expression of the zinc finger protein HKZNF-2 3 in a disease state.
  • the DNA sequence encoding zinc finger protein HKZNF-2 3 can be used to hybridize biopsy specimens to determine the expression of zinc finger protein HKZNF-2 3.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • Some or all of the polynucleotides of the present invention can be used as probes to be fixed on micro arrays or DNA chips (also known as “gene chips") for analyzing differential expression analysis of genes in tissues and genetic diagnosis.
  • Zinc finger protein HKZNF-23 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) amplification in vitro to detect the transcription product of zinc finger protein HKZNF-23.
  • Zinc finger protein HKZNF-23 mutations include point mutations, translocations, deletions, recombinations and any other abnormalities compared to the normal wild type zinc finger protein HKZNF-23 DNA sequence. Mutations can be detected using existing techniques such as Sou thern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nort Hern blotting and Wes tern 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.
  • the specific loci of each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data can be used to mark chromosome locations.
  • an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 1-35 bp) are prepared based on cDNA, and the sequences can be mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DM 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 cDNA clones to metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all 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 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 present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders authorize them to be administered to humans by government agencies that manufacture, use, or sell them.
  • 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.
  • the zinc finger protein HKZNF-23 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of zinc finger protein HKZNF-23 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.
  • RNA Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using the Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. Use Smart cDNA Cloning Kit (purchased from Clontech). The 0 fragment was inserted into the multiple cloning site of pBSK (+) vector (Clontech), and transformed into DH5cc. The bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • a comparison was performed with the public DNA sequence database (Genebank), and the cDNA sequence of one of the clones 0076cll was found to be new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the 0076cll clone contains a full-length cDNA of 3351bp (as shown in Seq IDN0: 1), and has a 213bp open reading frame (0RF) from 2095bp to 2736bp, encoding a new protein (such as Seq ID NO: 2).
  • the sequence of the zinc finger protein HKZNF-23 and the protein sequence encoded by the zinc finger protein of the present invention are
  • CDM was synthesized using fetal brain cell total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, PCR was performed using the following primers:
  • Primerl 5 ⁇ -GGGGATCTCAAAATGGCGGCCCCG- 3 '(SEQ ID NO: 3)
  • Primer2 5 "-TATTGACTTCTCTATTATCTTTGA-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 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l reaction volume containing 50 ol / L KC1, 10ramol / L Tris-CI, (pH8.5), 1.5mmol / L MgCl 2 , 200 ⁇ / L dNTP, lOpmol primer, 1U Taq DNA polymerase (C 1 on tech).
  • 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.
  • 3-act in is used as a positive control and template blank is used as a negative control.
  • the amplified product is purified using a QIAGEN kit, and the TA cloning kit is connected to a pCR vector (Invitrogen product).
  • DNA The sequence analysis results showed that the DM sequence of the PCR product was exactly the same as that of 1-3351bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of zinc finger protein HKZNF-23 gene expression
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-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 resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7.4) -5 x SSC- 5 x Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, the filters were washed in 1Q SSC-0.1% SDS at 55 Q 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 HKZNF-23
  • Primer3 5'- CCCGGATCCATGATACACTGTGAAGCTACTC- 3, (Seq ID No: 5)
  • Primer4 5'- CCCAAGCTTTTATCTGATGTATAATGAGTTC-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain BamHI and Hindlll digestion sites, respectively, followed by the coding sequences of the 5 'and 3' ends of the target gene, respectively.
  • the BamHI and Hindl 11 restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3).
  • the PCR reaction was performed using the pBS-0076cll plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-0076cll-containing plasmid in a total volume of 50 ⁇ l, primers Primer-3 and Primer-4 were lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles.
  • the amplified product and plasmid pET-28 (+) were double-digested with BamHI and Hindlll, respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into the colibacillus DH5 ⁇ by the calcium chloride method.
  • a peptide synthesizer (product of PE company) was used to synthesize the following zinc finger protein HKZNF-23-specific peptides: NH 2 -Met-Ile-His-Cys-Glu-Ala-Thr-His-Cys-Gly-Lys-Ile-Leu-Ser-His- C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43.
  • Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose.
  • 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 zinc finger protein HKZNF-23.

Abstract

L'invention concerne un nouveau polypeptide, une protéine à doigt de zinc HKZNF-23, 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 à doigt de zinc HKZNF-23.
PCT/CN2000/000326 1999-10-22 2000-10-16 Nouveau polypeptide, proteine a doigt de zinc hkznf-23, et un polynucleotide codant pour ce polypeptide WO2001030832A1 (fr)

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AU77691/00A AU7769100A (en) 1999-10-22 2000-10-16 A new polypeptide-zinc finger protein hkznf-23 and the polynucleotide encoding it

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CN99119817.4 1999-10-22
CN99119817 1999-10-22

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DNA, vol. 8, no. 6, 1989, pages 377 - 387 *
MAMM. GENOME, vol. 9, no. 6, 1998, pages 458 - 462 *

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