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WO2001070779A1 - Nouveau polypeptide, proteine humaine 12 de type cdc4, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 12 de type cdc4, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001070779A1
WO2001070779A1 PCT/CN2001/000157 CN0100157W WO0170779A1 WO 2001070779 A1 WO2001070779 A1 WO 2001070779A1 CN 0100157 W CN0100157 W CN 0100157W WO 0170779 A1 WO0170779 A1 WO 0170779A1
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Prior art keywords
polypeptide
polynucleotide
protein
human
cdc4
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU39107/01A priority Critical patent/AU3910701A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • 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
    • 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 new human CDC4 analogous protein 12 and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • the cell cycle is very orderly, following the order G1—S—G2—M—this is the result of the orderly expression of genes related to the progress of the cell cycle.
  • the genes involved in cell division are called cdc (cell division cycle gene).
  • the orderly expression of these genes is regulated by some checkpoints or control points in the cycle. The role of this point is to act as a regulatory pathway for the timing of cell cycle transitions. It ensures that key events in the cell cycle are completed with high accuracy. It is subject to a series of specific or specific environmental signals. At the molecular level, it is based on the response of some genes and their products to external signals.
  • Cell cycle checkpoints are commonly found in higher eukaryotes and yeast cells, such as yeast cells.
  • cdc3, cdc4, cdc8, and cdcl2 There is a family of cell division regulating genes in S. saccharophila, and four types have been found: cdc3, cdc4, cdc8, and cdcl2 (Nurse, P., P. Thur iaux, and L Nasmy th. 1976. Mol. & Gen. Genet. 146: 167- 178).
  • the cell division cycle regulatory gene (ce 11 di visi on eye 1 e gene, CDC4) has been cloned. It encodes a 141 amino acid peptide.
  • This protein is related to EF-chiral proteins (such as myosin light chain, calcium).
  • EF-chiral proteins such as myosin light chain, calcium
  • cdc4 protein can form a 200KD complex, and this complex can bind to UTP to play a certain role.
  • the protein encoded by the cell division cycle gene is a cell division cycle regulatory factor (CDC4).
  • CDC4 cell division cycle regulatory factor
  • the protein cloned from the cDNA library of rat 12-day embryo heart tissue has been researched ⁇
  • the division cycle regulatory protein CDC4 has a similar repeat structure to the betal subunit of the G protein. According to its highly conserved secondary structure, this repeat structure has a specific function: (DA Seq 1992; 3 (4): 213- 20)
  • cyclin-dependent kinase (CDK) inhibitor S 1 C 1 is cell cycle dependent.
  • SK '1 is stable in the early G1 phase to prevent the premature activation of CDK in S phase, and CDK and G1 cyclin ( cyclin) combined to generate an activated G1 phase CDK- cyclin complex, which phosphorylates S1C1, prompting the latter to be hydrolyzed through the CDC34 pathway, and then activated S phase CDK, which initiates DNA replication by phosphorylating some key substrates.
  • CDK cyclin-dependent kinase
  • the CDC4 gene product of Schizosaccharomyces cerevisiae is indispensable in the transition from the late G1 phase to the S phase of the cell cycle. Immunofluorescence assay has proved the localization of the CDC4 gene product in the nucleus. Previous studies have found that CI) It is an important component protein of the nuclear skeleton, but the specific function of the CDC4 gene product needs further study. (Biochem Biophys Res Commun 1990 Nov 15; 172 (3): 1324-30) The mutated cdc4 gene of the mitotic cell cycle will inhibit the initiation of nuclear DNA replication and the isolation of the spindle after replication. The cdc4 gene also plays an extremely important role in meiosis (Genetics 1977 May; 86 (1): 57-72)
  • SEL-10 also belongs to the CDC4 protein family. In the study of nematodes, SEL-10 was found to be a negative regulator of lin-12 / Notch regulatory signals. SEL-10 can increase the lin-12 / Notch regulatory signals stimulated by ubiquitin. Thus SEL-10 can regulate the activity of lin-12 / Notch and can indirectly regulate tumorigenesis: (Genes Dev 1997 Dec 1; 11 (23): 3182-93)
  • Presenilin genes include PS1 and PS2, whose mutations can cause Alzheimer's syndrome in humans. (Proc Natl Acad Sci U S A 1998 Dec 22; 95 (26): 15787-91)
  • the human CDC4-like protein 12 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need in the art to identify more of these processes
  • Human CDC4 is similar to protein 12, especially the amino acid sequence of this protein. Isolation of the new human CDC4-like protein 12 protein-encoding gene Identifying the role of this protein in health and disease states provides the basis. This protein may constitute a development disease
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human CDC4-like protein 1 2.
  • Another object of the present invention is to provide a method for producing human CDC4-like protein 12.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human CDC 4 -like protein 1 2.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the human CDC 4 analogous protein 1 2 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating a disease associated with a human CDC-like protein 1 2 abnormality.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 2, or a conservative variant, biologically active fragment, or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID D0: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • (C) A polynucleotide having at least 70% identity to a polynucleotide sequence of (a) or (b).
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 103-444 in SEQ ID NO: 1; and (b) a sequence having positions 1- in SEQ ID NO: 1 1 075-bit sequence.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human CDC4 analogous protein 12 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of human CDC4 similar protein 12 protein in vitro, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting a biological sample The amount or biological activity of a polypeptide of the invention.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention for the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human CDC4-like protein 12.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, which can be single-stranded or double-stranded, meaning the sense or antisense strand.
  • amino acid sequence refers to oligopeptides, peptides, polypeptides, or protein sequences and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bio activity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active means that natural, recombinant, or synthetic proteins and fragments thereof Ability to induce a specific immune response in a substance or cell and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human CDC4-like protein 12, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human CDC4-like protein 12.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human CDC4-like protein 12 when combined with human CDC4-like protein 12.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human CDC4-like protein 12.
  • Regular refers to a change in the function of human CDC4-like protein 12, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of human CDC4-like protein 12.
  • substantially pure means substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated.
  • Those skilled in the art can purify human CDC4-like protein 12 using standard protein purification techniques.
  • a substantially pure human CDC4-like protein 12 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of human CDC4-like protein 12 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences.
  • the percentage identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.).
  • the MEGALIGN program can compare two or more sequences based on different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0
  • the Clus ter method groups each group by checking the distance between all pairs. The sequences are arranged in clusters. The clusters are then assigned in pairs or groups.
  • Two amino acid sequences such as The percent identity between column A and sequence B is calculated by the following formula: The number of residues matching between sequence A and sequence X X 100 The number of residues in sequence A-the number of residues in sequence A-the interval in sequence B The number of residues can also be determined by the Cluster method or by methods known in the art such as Jotun He in.
  • the percentage identity of a nucleic acid sequence Hein J., (1990) Methods in emzumology 183: 625-645
  • similarity It refers to the degree of the same or conservative substitution of amino acid residues at corresponding positions when the amino acid sequences are aligned.
  • Amino acids used for conservative substitutions may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine:
  • the uncharged head group has Similar hydrophilic amino acids may include leucine, isoleucine and valine: glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine
  • Antisense refers to a nucleotide sequence that is complementary to a specific DNA or RNA sequence: "Antisense strand” refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to human CDC4-like protein 12 epitopes.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state.
  • isolated human CDC4-like protein 12 means that human CDC4-like protein 1 2 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 CDC4-like protein 12 using standard protein purification techniques.
  • Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of human CDC4-like protein 12 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a human CDC4 analogous protein 12, which basically consists of the amino acid sequence shown in SEQ II) 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 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).
  • the polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the present invention may also include or exclude initial methionine residues; the present invention also includes fragments, derivatives, and analogs of human CDC4 analogous protein 12.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human CIK '4 analogous protein 12 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 A amino acid residues is replaced by another group to include a substituent; or (III) such One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • an additional amino acid sequence is fused into the mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protea
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 1075 bases, and its open reading frame 103-444 encodes 113 amino acids.
  • the polynucleotide of the present invention can be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 The same or degenerate variants.
  • 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; "J: the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) ) And non-coding sequences.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) added during hybridization Use a denaturant, such as 503 ⁇ 4 (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% It is more preferable that hybridization occurs at 97% or more, and 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 human CDC4-like protein 12.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human CDC4-like protein 12 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) Isolation of double-stranded DNA from genomic DNA Sequence; 2) chemically synthesize a DM sequence to obtain double-stranded DNA of the polypeptide.
  • genomic DM 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 raRNA from donor cells that overexpress the gene and perform reverse transcription to form a 'granule or phage cDNA library. There are many mature techniques for extracting 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 cDNA libraries are also available, such as Clontech's non-cDNA library. When polymerase reaction technology is used in combination, even very few expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determining the level of human CDC4-like protein 12 transcripts; (4) The protein product of a gene is detected by immunological techniques or by measuring biological activity. The above methods can be used alone or in combination:
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human CDC4-like protein 12 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), etc.—
  • a method using PCR 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 determined based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA segment can be isolated and purified by conventional methods such as gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention obtained as described above, or various DNA fragments can be conventional methods such as dideoxy chain termination ( Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a vector or a direct use of the vector of the present invention.
  • a polynucleotide sequence encoding a human CDC4-like protein 12 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.
  • 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.
  • 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 DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include late stage one at the origin of replication: the regular SV40 enhancer of 100 to 270 base pairs, polyoma enhancers at the late side of the origin of replication, and adenoviral enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide the phenotypic traits of the selected transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli, etc.
  • a polynucleotide encoding human CDC4 similar protein 12 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host cells 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.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast: plant cells: insect cells such as fly S 2 or Sf 9
  • animal cells such as CH0, COS, or Bowes s melanoma Cells etc.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with CaC l 2 method, used are well known in the art ho step. The alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human CDC4-like protein 12 (Scence, 1984; 224: 1 4 31). Generally, the following steps are taken:
  • 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, or expressed on a cell membrane, or separated, and must be extracellular. If desired, recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • FIG. 1 is a gene chip expression profile ratio of human CDC4 analogous protein 12 and human CDC4 analogous protein 1 3 of the present invention Compare the figure.
  • the upper graph is a graph of the expression profile of human CDC4-like protein 12, and the lower sequence is the graph of the expression profile of human CDC4-like protein 13.
  • Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of isolated human CDC4 similar protein 12 ; 12kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • 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.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragments into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5 ⁇ .
  • the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0434a09 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the 0434a09 clone contains a full-length cDNA of 1075bp (as shown in Seq IDNO: 1), and has a 341bp open reading frame (0RF) from 103bp to 444bp, encoding a new protein (such as Seq ID NO: 2).
  • This clone pBS_0434a09 and the encoded protein was named human CDC4-like protein 12.
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:
  • Primer 1 5 _ GATTGCGTAGCGAAGCCCCCGGCG- 3 '(SEQ ID NO: 3)
  • Primer2 5'- TTTTCAATACACTTTAATAAAATA-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at Ibp at the 5 ′ end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l of KC1, 10 mmol / L Tris- in a reaction volume of 50 ⁇ 1 CI, (pH 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpraol primer, 1U Taq DNA enzyme (product of Clontech). Reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles according to the conditions listed below: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. At the same time, ⁇ -ac t was set during RT-PCR. In is the positive control and the template blank is the negative control.
  • Electrophoresis was performed on a 1.2% agarose gel containing 20 ⁇ ⁇ RNA on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7. Q)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • A- 32 P dATP was used to prepare ⁇ P-labeled DNA probes by random primer method.
  • the DNA probe used was the 'coding region' (103bp to 444bp) of the human CDC4-like protein 12 coding region amplified by PCR shown in FIG. 1.
  • a 32P-labeled probe (about 2 x 10 6 cpffl / 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 (pH 7.4) -5 x SSC-5 x Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in lx SSC — 0.1% SDS at 55 ° C. for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human CDC4 analogous protein 12
  • Primer 3 5'-CCCCATATGATGGGTAAAGCCAGGAGGAAGCCC-3 '(Seq ID No: 5)
  • Primer4 5'_CATGGATCCCTACCACCCTGATCTATTTGGCTT_3, (Seq ID No: 6)
  • the 5 ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5, 5 and 3 'ends of the target gene, Ndel and BamHI
  • the restriction site corresponds to a selective endonuclease site on the expression vector plasmid pET-28b (+) (Nova gen, Cat. No. 69865.3).
  • the PCR reaction was performed using the P BS-0434a09 plasmid containing the full-length target group g as a template.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ 1 contains 10 pg of pBS_0434a09 plasmid, Prime I-3 and Primer-4 points; j is lpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68. C 2 min. 25 in total Loop. Nde I and BaraH I were used to double-digest the amplified product and plasmid ET-28 (+), respectively. Large fragments were recovered and ligated with T4 ligase. The ligated product was transformed with colibacillus DH5cx by the calcium chloride method.
  • the positive clones were selected by colony PCR method and sequenced. Select positive clones with correct sequence (pET-0434a09). Transform the recombinant plasmid into Escherichia coli BL21 (DE3) plySs (Novagen) with calcium chloride method in LB liquid containing kanamycin (final concentration 30 ⁇ g / nil) In the medium, the host strain BL21 (pET-0434a09) was cultured at 37 ° C. to a logarithmic growth phase, IPTG was added to a final concentration of lnimol / L, and the culture was continued for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge was used which could bind 6 histidine (6His-Tag).
  • a peptide synthesizer (product of PE company) was used to synthesize the following human CDC4-like protein 12-specific peptides:
  • a titer plate coated with 15 ⁇ g / ml bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum.
  • Protein A-Sepharose was used to separate total IgG from antibody-positive home-immunized serum.
  • the peptide was bound to a cyanogen bromide-activated Sepharose 4B column, and anti-peptide antibodies were isolated from total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibody specifically binds to human CDC4-like protein 12.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is identified whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can also be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues or Whether the expression in pathological tissue cells is abnormal.
  • the purpose of this example is to select a suitable oligonucleoside from the polynucleotide SEQ ID NO: 1 of the present invention
  • the acid fragment is used as a hybridization probe, and the membrane hybridization method is used to identify whether some tissues contain the polynucleotide sequence of the present invention or a homologous polynucleotide sequence.
  • Filter hybridization methods include dot blotting, Southern blotting, Nor thern blotting, and copying methods. They are all based on the same method of immobilizing the polynucleotide sample to be tested on the filter and then using basically the same step hybridization.
  • the Xiangpin-fixed membrane is first pre-hybridized with a probe-free hybridization buffer so that the non-specific binding site of the sample on the membrane is saturated with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washes.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements Region for homology comparison, if the homology with the non-target molecular region is greater than 85% or more than 15 consecutive bases are identical, the primary probe should generally not
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffered saline
  • step 14 Resuspend the DNA pellet in a small volume of TE or water. Low-speed vortexing or pipetting, with a dropper, while gradually increasing the TE, mixed until fully dissolved DNA, every 1- 5 ⁇ 10 6 cells extracted about plus lul.
  • steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • the sample membrane was placed in a plastic bag, and 3- 10 mg of prehybridization solution (10xDenhardt's; 6xSSC, 0. lrag / ml) was added.
  • CT DNA (calf thymus DNA).
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data are compared and analyzed on a carrier such as silicon, and then fluorescence detection and computer software are used to achieve fast, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, see DeRisi, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And Helle, RA, Schema, M. Chai, A., Shalom, I)., (1997) PNAS 94: 2150-2155. (A)
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and a Cartesian 7500 spotter (purchased from Cartesian, USA) was used to spot the glass medium. The distance is 280 ⁇ m. The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method ⁇ step has been variously reported in the literature. The post-sampling processing steps of this embodiment are:
  • Probes from the above two types of tissues were hybridized with the chip in a UniHyb TM Hybridization Solution (purchased from TeieChem) for 16 hours, washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000.
  • Scanner purchased from General Scanning Company, USA
  • Imagene software Biodiscovery, USA
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour.
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • the polypeptide of the present invention (human CDC4-like protein 13) is a cell division cycle regulating factor, which is very important for ensuring the orderly operation of the cell cycle. If this regulatory pathway is abnormal, the cycle cannot function properly and the key events of the cycle are lost Loyalty, which results in genetic performance disorders, abnormal proliferation and differentiation, and cell canceration, and even cell death.
  • the polypeptide of the present invention also has some important functions.
  • the cdc4 gene also plays an extremely important role in meiosis;
  • the CDC4 gene product is an important component protein of the nuclear skeleton;
  • the polypeptide of the present invention can regulate lin-12 / Notch And can indirectly regulate the occurrence of tumors;
  • the polypeptide of the present invention can regulate the level of the presenilin gene, thereby having an effect on human Alzheimer's syndrome.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human CDC4-like protein 12.
  • Agonists enhance biological functions such as human CDC4-like protein 12 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human CDC4-like protein 12 can be cultured with labeled human CDC4-like protein 12 in the presence of drugs. The ability of the drug to increase or suppress this interaction was then determined.
  • Antagonists of human CDC4-like protein 12 included screened antibodies, compounds, receptor deletions, and the like.
  • Antagonists of human CDC4-like protein 12 can bind to human CDC4-like protein 12 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide such that the polypeptide cannot perform biological functions.
  • human CDC4 analogous protein 12 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between human CDC4 analogous protein 12 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 CDC4-like protein 12 may It is obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, generally, 12 molecules of human CDC4-like protein should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as a precursor.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human CDC4 analogous protein 12 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and Fab expression library-derived fragments.
  • Polyclonal antibodies can be produced by injecting human CDC4 analogous protein 12 directly into immunized animals (eg, home, mouse, rat, etc.).
  • immunized animals eg, home, mouse, rat, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to two Freund's adjuvants.
  • Techniques for preparing monoclonal antibodies to human CDC4-like protein 12 include, but are not limited to, hybridoma technology (Kohler and Milstein, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridization Tumor technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morr is on etal, PNAS, 1985, 81: 685 1) 0 and f single-chain antibody production techniques (US Pa t No. 4946778) can also be used to produce single-chain antibodies against human CDC4 similar to white 12.
  • Anti-human CDC4-like protein 12 antibodies can be used in immunohistochemical techniques to detect human CDC4-like protein 12 in biopsies.
  • Monoclonal antibodies that bind to human CDC4-like protein 12 can also be labeled with radioisotopes, and their location and distribution can be tracked in vivo.
  • 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 CDC4 is similar to white 12 and high-affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, legumin, etc.).
  • a common method is to use a sulfhydryl crosslinker such as SPDP to attack the ⁇ of an antibody and bind the toxin to the antibody through the exchange of disulfide bonds.
  • SPDP sulfhydryl crosslinker
  • This hybrid antibody can be used to kill human CDC-like protein 12 positive cells.
  • the antibodies of the present invention can be used to treat or prevent human CDC4-like protein 12-related diseases. Administration of an appropriate dose of the antibody can stimulate or block human CDC4-like protein 12 production or activity.
  • the present invention also relates to diagnostic assays for quantitative and localized detection of human CDC4-like protein 12 levels: These tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the levels of human CDC4-like protein 12 measured in the test can be used to explain the importance of human CDC4-like protein 12 in various diseases and to diagnose diseases in which human CDC4-like protein 12 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis, for example, the polypeptide can be physical, chemical or enzymatic: Specific cleavage and one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, preferably mass spectrometry.
  • Polynucleotides encoding human CDC4-like protein 12 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of human CDC4-like protein 12.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human CDC4-like protein 12 to inhibit endogenous human CDC4-like protein 12 activity :
  • a mutated human CDC4-like protein 12 can be Shortened human CDC4 analogous protein 12 lacking the 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 human CDC4-like protein 12.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. are available Methods for transferring a polynucleotide encoding human CDC4-like protein 12 into a cell to construct a recombinant viral vector carrying a polynucleotide encoding human CDC4-like protein 12 can be found in existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding human CDC4-like protein 12 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human CDC4-like protein 12 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 using any existing RNA or DNA synthesis technology, such as the solid-phase phosphoramidite chemical synthesis method for oligonucleotide synthesis.
  • 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 vector's RNA polymerase promoter.
  • it can be modified in a variety of ways, increasing the sequence length on both sides, and the ribonucleosides should be bonded using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human CDC4-like protein 12 can be used for the diagnosis of diseases related to human CDC4-like protein 12.
  • the polynucleotide encoding human CDC4-like protein 12 can be used to detect the expression of human CDC4-like protein 12 or the abnormal expression of human CDC4-like protein 12 in a disease state.
  • the DNA sequence encoding human CDC4-like protein 12 can be used to hybridize biopsy specimens to determine the expression of human CDC4-like protein 12.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, etc .: These technical methods are publicly available mature technologies, and related kits are available commercially.
  • the hair Some or all of the polynucleotides can be used as probes to be fixed on a microarray or a DNA chip (also known as a "gene chip"), and used to analyze the difference table analysis and basic diagnosis of genes in tissues:
  • Human CDC4-like protein 12 specific primers can also be used to detect human CDC4-like protein 12 transcripts by RNA-polymerase chain reaction (RT-PCR) in vitro amplification.
  • Detection of mutations in the human CDC4-like protein 12 gene can also be used to diagnose human CDC4-like protein 12-related diseases.
  • Human CDC4-like protein 12 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human CDC4-like protein 12 DNA sequence.
  • Existing techniques such as Southern blotting, DNA sequence analysis, PCR, and in situ hybridization: mutations.
  • mutations may affect the expression of proteins, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • the specific loci of each gene on chromosome 2 need to be identified.
  • only few chromosomal markers based on actual sequence data are available for marking chromosome positions.
  • the important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared from the cDNA, and the sequences can be located on the chromosomes. These primers are then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human genes corresponding to the primers will produce amplified slices:
  • the somatic hybrid cell PCR mapping method is a fast method for locating DNA to a specific chromosome.
  • oligonucleotide primers of the present invention by a similar method, a group of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sub- Positioning.
  • 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 build chromosome-specific c-shiphouses.
  • Fluorescent in situ hybridization of cDNA clones to metaphase chromosomes allows precise chromosomal localization in a single step.
  • FISH Fluorescent in situ hybridization
  • this sequence can be correlated with the genetic map data at the physical location of chromosome two. 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 at A mutation is observed in some or all diseased individuals, and the mutation is observed in any normal individual-then the mutation may be the cause of the disease. Comparing diseased 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 PCR based on c DNA sequences. 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 Graph resolution and each 20k b 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 CDC4 analogous protein 12 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human CDC4-like protein 12 to be administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine 12 de type CDC4, 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 humaine 12 de type CDC4.
PCT/CN2001/000157 2000-03-07 2001-02-26 Nouveau polypeptide, proteine humaine 12 de type cdc4, et polynucleotide codant pour ce polypeptide Ceased WO2001070779A1 (fr)

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CN00111935A CN1312284A (zh) 2000-03-07 2000-03-07 一种新的多肽——人cdc4类似蛋白12和编码这种多肽的多核苷酸
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993023571A1 (fr) * 1992-05-12 1993-11-25 Fred Hutchinson Cancer Research Center Genes points de controle du cycle cellulaire humain
WO1997041238A1 (fr) * 1996-05-02 1997-11-06 Pharmacia & Upjohn Company Macromolecules catalytiques presentant une activite semblable a celle de la cdc25b
US5871957A (en) * 1984-05-25 1999-02-16 Zymogenetics, Inc. Stable DNA constructs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5871957A (en) * 1984-05-25 1999-02-16 Zymogenetics, Inc. Stable DNA constructs
WO1993023571A1 (fr) * 1992-05-12 1993-11-25 Fred Hutchinson Cancer Research Center Genes points de controle du cycle cellulaire humain
WO1997041238A1 (fr) * 1996-05-02 1997-11-06 Pharmacia & Upjohn Company Macromolecules catalytiques presentant une activite semblable a celle de la cdc25b

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