WO2001029228A1 - Nouveau polypeptide, caseine kinase humaine 48, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, caseine kinase humaine 48, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001029228A1
WO2001029228A1 PCT/CN2000/000330 CN0000330W WO0129228A1 WO 2001029228 A1 WO2001029228 A1 WO 2001029228A1 CN 0000330 W CN0000330 W CN 0000330W WO 0129228 A1 WO0129228 A1 WO 0129228A1
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polypeptide
polynucleotide
casein kinase
human casein
seq
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PCT/CN2000/000330
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Shanghai Bio Road Gene Development Ltd.
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Priority to AU78991/00A priority Critical patent/AU7899100A/en
Publication of WO2001029228A1 publication Critical patent/WO2001029228A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, human casein kinase 48, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing the polynucleotide and polypeptide.
  • Casein kinase 1 is a monomeric form of a protein kinase that favors acidic proteins. Substrates are widely present in eukaryotic cells. Casein kinase was originally thought to be a serine kinase. Recently, however, it has been reported that the CKI of several yeasts can phosphorylate tyrosine on their own protein, which indicates that the protein has dual specific enzyme activity.
  • CKI is widely distributed in various tissues in the body and localized differently in different compartments of the cell, which implies that CKI has important regulatory functions.
  • a member of the CKI is combined with the cytoplasmic corpus callosum, which has the function of cell cycle regulation.
  • CKI can phosphorylate the Ser-10 site of glycogen synthase protein in rabbit muscle tissue.
  • This site is believed to be related to the regulation of hormone levels in the body; the activity of SV40 large T antigen is also regulated by CKI phosphorylation modification; the N-terminus of P53 protein is also modified by CKI phosphorylation, but the physiological role of this process is still Not sure.
  • CKI actually represents a multi-gene family and is widely present in eukaryotic cells. Rowles et al first described the cDNA members of the CKI family. He cloned two full-length CKI genes from a bovine brain cDNA library, which encode 37600 and 38700, respectively. Dalton's protein, he named it CKIa and ⁇ ⁇ .
  • CKI Y 1 and CKI Y 3 genes were transferred into yeast for expression, respectively, and yeast morphology and growth returned to normal, which proves that the functions of some mammalian CKI genes and yeast's corresponding CKI genes can be complementary.
  • the CU gene we cloned from the human fetal brain library was a homolog of the mouse CKI Y 1 gene, and we named it ⁇ -CKI Y 1.
  • the H-CKI Y 1 protein belongs to the large family of CKI proteins and has Ser / Tyr and Tyr protein kinase conserved regions. The first conserved region is located at the N-terminus, and is rich in glycine near the extremely conserved cysteine residues. This region binds to ATP. The second region is located in the middle of the protein and has a Very conserved glutamate residues, this region is necessary for the catalytic activity of the enzyme. (Hanks sk, Hunter T. et al).
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a method for producing human casein kinase 48.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, human casein kinase 48.
  • Another object of the present invention is to provide analog compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, human casein kinase 48.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human casein kinase 48.
  • a novel isolated human casein kinase 48 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 Active fragments, or active derivatives, analogs thereof.
  • 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 95 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human casein kinase 48; (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 199-1515 in SEQ ID NO: 1; and (b) a sequence having 1-1754 in SEQ ID NO: 1 Sequence of bits.
  • FIG. 1 is a comparison diagram of amino acid sequence homology between human casein kinase 48 of the present invention and mouse CKI Y 1.
  • the upper sequence is human casein kinase 48, and the lower sequence is mouse ⁇ 1.
  • 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 isolated human casein kinase 48.
  • 48.2 kDa 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 human casein kinase 48 means that human casein kinase 48 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human casein kinase 48 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the human casein kinase 48 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human casein kinase 48, 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 human casein kinase 48.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human casein kinase 48 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) 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 mature peptide Fusion with another compound such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol
  • a polypeptide sequence such as a leader sequence or a secretion sequence formed by fusing additional amino acid sequences into a mature polypeptide Or the sequence used to purify this polypeptide or protease sequence
  • 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 CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 1754 bases in length and its open reading frame (199-1515) encodes 438 amino acids.
  • this polypeptide has 93% homology with the mouse ⁇ , and that the polypeptide has four conservative bases of the CKI gene family. It can be inferred that the new human H-CKI Y 1 Has a similar structure and function of the CKI gene family.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cD, 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 D NO: 2 but different from the coding region sequence shown in SEQ ID D 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 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 invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) in the lower Hybridization and elution at ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60'C; or (2) adding a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% less Bovine serum / 0.1% Ficoll, 42'C, etc .; or (3) hybridization occurs only when the identity between the two sequences is at least 95% or more, and more preferably 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 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human casein kinase 48.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human casein kinase 48 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) separating the double-stranded DM 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 DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Various methods have been used to extract mRNA, 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 cDM 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 D-RNA hybrids; (2) the presence or absence of marker gene functions; (3) determining the level of human casein kinase 48 transcripts; (4) Detection of gene-expressed protein products by immunological techniques or determination of biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • probes are typically 2,000 nucleotides in length Within 1,000 nucleotides is preferred.
  • the probe used herein is usually a D sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human casein kinase 48 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).
  • 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 DM fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the 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 genetically engineered using the vector of the present invention or directly using the human casein kinase 48 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding human casein kinase 48 may be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors 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.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human casein kinase 48 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the D 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 and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for D expression, usually about 10 to 300 base pairs that act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs at a later stage of the origin of replication, polyoma enhancers and adenovirus enhancers at the late side of the origin of replication, and the like.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human casein kinase 48 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 a 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 include: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as S2 or Sf9; animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be harvested after exponential growth phase, with (: & (: Treatment Step 12, the used well known in the art alternative is to use MgCl 2
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, Liposome packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human casein kinase 48 by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention 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 invention also provides methods for screening compounds to identify agents that increase (agonist) or inhibit (antagonist) human casein kinase 48.
  • Agonists enhance human casein kinase 48 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 human casein kinase 48 can be cultured together with labeled human casein kinase 48 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human casein kinase 48 include antibodies, compounds, receptor deletions and analogs that have been screened. Antagonists of human casein kinase 48 can bind to human casein kinase 48 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.
  • human casein kinase 48 When screening compounds as antagonists, human casein kinase 48 can be added to bioanalytical assays to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between human casein kinase 48 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 human casein kinase 48 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 human casein kinase 48 molecule should generally be labeled.
  • the present invention provides polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. To produce antibodies. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against human casein kinase 48 epitopes. 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 human casein kinase 48 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human casein kinase 48 include, but are not limited to, hybridoma technology (ohl er and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma Technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that combine human constant regions with non-human variable regions can be produced using existing techniques (Morr et al, PNAS, 1985, 81: 6851) ⁇ and existing techniques for producing single-chain antibodies (US Pa t No. 4946778) can also be used to produce single chain antibodies against human casein kinase 48.
  • Anti-human casein kinase 48 antibodies can be used in immunohistochemical techniques to detect human casein kinase 48 in biopsy specimens.
  • Monoclonal antibodies that bind to human casein kinase 48 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human casein kinase 48 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human casein kinase 48-positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases associated with human casein kinase 48. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human casein kinase 48.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human casein kinase 48 levels.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of human casein kinase 48 detected in the test can be used to explain the importance of human casein kinase 48 in various diseases and to diagnose diseases in which human casein kinase 48 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.
  • human casein kinase 48 can also be used for a variety of therapeutic purposes. Gene therapy technology It can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human casein kinase 48.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human casein kinase 48 to inhibit endogenous human casein kinase 48 activity.
  • a variant human casein kinase 48 may be a shortened human casein kinase 48 lacking a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human casein kinase 48.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human casein kinase 48 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human casein kinase 48 can be found in existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human casein kinase 48 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 human casein kinase 48 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific R. 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 using any existing RNA or DM synthesis techniques, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human casein kinase 48 is useful for the diagnosis of diseases related to human casein kinase 48.
  • the polynucleotide encoding human casein kinase 48 can be used to detect the expression of human casein kinase 48 or the abnormal expression of human casein kinase 48 in a disease state.
  • the DNA sequence encoding human casein kinase 48 can be used to hybridize biopsy specimens to determine the expression of human casein kinase 48.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human casein can also be detected using RNA-polymerase chain reaction (RT-PCR) in vitro with human casein kinase 48-specific primers Transcription product of kinase 48.
  • RT-PCR RNA-polymerase chain reaction
  • Human casein kinase 48 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human casein kinase 48 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, so Northern 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.
  • PCR primers (preferably 15-35bp) are prepared based on the cDNA, and the sequences can be located on the 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 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 cDNA clones with 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 differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, as seen at the chromosomal level or detectable by cDNA sequence-based PCR Deletion or translocation. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human casein kinase 48 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human casein kinase 48 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.
  • the determined cDNA sequence was compared with the existing public D sequence database (Genebank), and one of the clones was found.
  • the cDNA sequence of 0180f02 is new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the results show that the full-length cDNA contained in the 0180f02 clone is 1754bp (as shown in Seq IDN0: 1), and there is a 1317bp open reading frame (0RF) from 199bp to 1515bp, which encodes a new protein (such as Seq ID NO: 2).
  • Example 2 Homologous search of cDNA clones
  • the sequence of the human casein kinase 48 of the present invention and the protein sequence encoded by the casein kinase 48 were subjected to the Blast program.
  • 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:
  • Primer 1 5'-GACCTAGTGACTACATTTCCTACG-3 '(SEQ ID NO: 3)
  • Primer2 5'- CAAGAAAGGTAAGACATTTTATTT- 3, (SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 'end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KC1, 10 mmol / L Tris-Cl, (pH 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ raol / L dNTP, lOpmol primer, 1U in a reaction volume of 50 ⁇ 1 Taq DNA polymer (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min 0 ⁇ -act in was set as positive at RT-PCR Controls and template blanks are negative controls.
  • Amplification products were purified using a QIAGEN kit and ligated to a pCR vector using a TA cloning kit (Unvitrogen product).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 1754bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human casein kinase 48 gene expression:
  • This method includes acid sulfur Guanidinium cyanocyanate-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. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA was prepared by c- 32 P dATP by random primer method.
  • the D probe used was the PCR amplified human casein kinase 48 coding region sequence (199bp to 1515bp) shown in FIG. 1.
  • a 32P-labeled probe (about 2 x 10 6 cpra / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25m KH 2 P0 4 (pH7.4)-5 x SSC- 5 x Denhardt's solution and 200 yg / ml salmon sperm DNA. After hybridization, the filters were 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 human casein kinase 48
  • Primer3 5'-CCCGGATCCATGGACCATCCTAGTAGGGAAAAGG-3 '(Seq ID No: 5)
  • Primer 4 5'-CCCAAGCTTCCAATACACTTGAAATTGACATTCA-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain BamHI and Hindlll digestion sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the restriction sites of BamHI and Hindlll correspond to the selectivity on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • the PCR reaction was performed using pBS-0180f02 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ l containing 10 pg of pBS-0180f02 plasmid, 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 Hindi 11, respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed into E.
  • the host bacteria BL21 (pET-0180f 02) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 ol / L, continue to cultivate for 5 hours.
  • the bacterial cells were collected by centrifugation, and the supernatant was collected by centrifugation, and the supernatant was collected by centrifugation.
  • An affinity column His Bind capable of binding to 6 histidines (6His-Ta g ) was used.
  • Quick Cartr idge (Novagen) was chromatographed to obtain purified human casein kinase 48.
  • the following peptides specific to human casein kinase 48 were synthesized using a peptide synthesizer (product of PE): NH 2 -Met-Asp-Hi s-Pro-Ser-Arg-Glu-Lys-Asp-Glu-Arg-Gln- Arg-Thr-Thr-COOH (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 4 mg of the i-cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once. ⁇ Using a 15 g / ml bovine serum albumin peptide complex-coated titer plate 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 human casein kinase 48.

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Abstract

L'invention concerne un nouveau polypeptide, une caséine kinase humaine 48, 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'agoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la caséine kinase humaine 48.
PCT/CN2000/000330 1999-10-18 2000-10-16 Nouveau polypeptide, caseine kinase humaine 48, et polynucleotide codant pour ce polypeptide WO2001029228A1 (fr)

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AU78991/00A AU7899100A (en) 1999-10-18 2000-10-16 A novel polypeptide, a human casein kinase 48 and the polynucleotide encoding the polypeptide

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6730491B2 (en) 2000-02-29 2004-05-04 Millennium Pharmaceuticals, Inc. 2504, 15977, and 14760, novel protein kinase family members and uses therefor
US7402653B2 (en) 2001-07-13 2008-07-22 Cms Peptides Patent Holding Company Limited Biologically active peptides

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019993A1 (fr) * 1994-01-21 1995-07-27 The Salk Institute For Biological Studies Proteine-kinases
US5897860A (en) * 1995-02-03 1999-04-27 Korea Institute Of Science Technology Method for controlling bleeding and microbial infections by administering thrombin, casein kinase II, and sphingosine
WO1999043811A1 (fr) * 1998-02-25 1999-09-02 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Adn complementaires codant un gene bog (b5t-over-expressed gene) et son produit proteinique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019993A1 (fr) * 1994-01-21 1995-07-27 The Salk Institute For Biological Studies Proteine-kinases
US5897860A (en) * 1995-02-03 1999-04-27 Korea Institute Of Science Technology Method for controlling bleeding and microbial infections by administering thrombin, casein kinase II, and sphingosine
WO1999043811A1 (fr) * 1998-02-25 1999-09-02 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Adn complementaires codant un gene bog (b5t-over-expressed gene) et son produit proteinique

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6730491B2 (en) 2000-02-29 2004-05-04 Millennium Pharmaceuticals, Inc. 2504, 15977, and 14760, novel protein kinase family members and uses therefor
US7402653B2 (en) 2001-07-13 2008-07-22 Cms Peptides Patent Holding Company Limited Biologically active peptides
US7491689B2 (en) 2001-07-13 2009-02-17 Cms Peptides Patent Holding Company Limited Compositions comprising the biologically active peptide YSL
US7696152B2 (en) 2001-07-13 2010-04-13 CMS Peptides Patent Holdings Company Limited Biologically active peptide consisting of tyrosyl-seryl-leucine (YSL)
US7718768B2 (en) 2001-07-13 2010-05-18 Cms Peptides Patent Holding Company Limited Biologically active peptide PTTKTYFPHF

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