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WO2001060855A1 - Nouvelle proteine humaine associee a la regulation du cycle cellulaire et sa sequence de codage - Google Patents

Nouvelle proteine humaine associee a la regulation du cycle cellulaire et sa sequence de codage Download PDF

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Publication number
WO2001060855A1
WO2001060855A1 PCT/CN2001/000121 CN0100121W WO0160855A1 WO 2001060855 A1 WO2001060855 A1 WO 2001060855A1 CN 0100121 W CN0100121 W CN 0100121W WO 0160855 A1 WO0160855 A1 WO 0160855A1
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clg
protein
polypeptide
human
polynucleotide
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PCT/CN2001/000121
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English (en)
Chinese (zh)
Inventor
Jianren Gu
Shengli Yang
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Shanghai Cancer Institute
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Priority to CNB018049176A priority Critical patent/CN1160370C/zh
Priority to AU2001233587A priority patent/AU2001233587A1/en
Publication of WO2001060855A1 publication Critical patent/WO2001060855A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • 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, and in particular, the present invention relates to a novel polynucleotide encoding a human cell cycle control related gene (crn-ike gen ', CLG) protein with cancer suppressing function, and the polynucleotide encoding Of peptides.
  • the invention also relates to the use and preparation of such polynucleotides and polypeptides.
  • Cell division and proliferation are one of the basic characteristics of cell life activity. During the growth and development of the human body and other multicellular organisms, the increase in the number of cells, the renewal of senescent and dead cells, and the continuation of life all need to be accomplished through cell proliferation.
  • the cell proliferation cycle is the process by which cells achieve cell growth and proliferation through a series of intracellular events. There is a strict sequence between each event. Its regulatory mechanism involves multiple levels and multiple factors, not only involving growth factors and cyclic nucleosides. Acids, hormones, proto-oncogenes, etc., are more importantly controlled by the cell cycle regulatory system, such as cell division cycle genes, cyclins, cyclin-dependent kinases and their inhibitory proteins.
  • the regulation of the cell cycle is a core issue in the research of cell biology. It involves the orderly development of embryonic cells, the normal proliferation of body cells, the regeneration and differentiation of cells, and the aging and apoptosis of cells. Normal cell cycle must be strictly and orderly regulated. Abnormal cell cycle regulation will cause cells to mutate, deform or become cancerous.
  • Cancer is one of the main diseases that endanger human health. In order to effectively treat and prevent tumors, people have paid more and more attention to gene therapy of tumors. Therefore, there is an urgent need in the art to develop and study human proteins and their agonists / inhibitors with cancer suppressing functions, especially proteins involved in cell cycle regulation.
  • the object of the present invention is to provide a new class of human protein polypeptides with tumor suppressing function, as well as fragments, analogs and derivatives thereof.
  • Another object of the invention is to provide polynucleotides encoding these polypeptides.
  • Another object of the present invention is to provide a method for producing these polypeptides and the use of the polypeptide and coding sequence.
  • a novel isolated protein polypeptide having a tumor suppressing function which comprises a polypeptide having the amino acid sequence of SEQ ID NO: 2; or a conservative variant polypeptide thereof, or an active fragment thereof, or an activity thereof derivative.
  • the polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
  • an isolated polynucleotide which comprises a nucleotide sequence that is at least 85% identical to a nucleotide sequence selected from the group consisting of: (A) a polynucleotide encoding the above-mentioned CLG protein polypeptide; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polypeptide encoded by the polynucleotide has the amino acid sequence of SEQ ID NO: 2. More preferably, the sequence of the polynucleotide is selected from the group consisting of the coding region sequence of SEQ ID NO: 3 (22-2586 positions) or full-length sequences (1-2659 positions).
  • a method for preparing a polypeptide having CLG protein activity comprises: (a) culturing the transformed or transduced host cell under conditions suitable for expressing the CLG protein; (b) ) A polypeptide having CLG protein activity is isolated from the culture.
  • an antibody that specifically binds to the CLG protein polypeptide described above is provided.
  • Nucleic acid molecules that can be used for detection are also provided, which contain 10-800 consecutive nucleotides in the above-mentioned polynucleotides.
  • a pharmaceutical composition which contains a safe and effective amount of the CLG protein polypeptide of the present invention and a pharmaceutically acceptable carrier.
  • These pharmaceutical compositions can treat diseases such as cancer and abnormal cell proliferation.
  • Figure 1 is the RNA expression profile of CLG.
  • Figure 2 shows the inhibitory effect of the full-length CLG cDNA on the colony formation of human hepatoma cell line S 7721.
  • FIG. 2A is the vector control (pCMV-Scr ipt) with 56 colonies;
  • Figure 2B is CLG (pCMV-Script / CLG) with only 3 colonies.
  • Figure 3 is a photograph of a paraffin section of tumor tissue formed by CLG-transfected S-deleted C-7721 cells inoculated in nude mice.
  • Figure 3A shows necrotic foci, and more positive apoptotic cells can be seen;
  • Figure 3B shows non-necrotic areas, and scattered apoptotic cells can be seen.
  • Figure 4 shows the "ladder" DNA electrophoresis map after CLG gene transfection.
  • the lanes are: 1. Transfected empty vector; 2. Transfected p53; 3. Transfected CLG; 4. Untransfected control point; 5. Molecular weight standard.
  • FIG. 5 SDS-PAGE electrophoresis of CLG protein expression.
  • each lane is: 1. protein molecular weight standard; 2. ultrasonic supernatant of pET32a-CLG bacteria; 3. ultrasonic supernatant of pET32a bacteria; 4. ultrasonic precipitation of pET32a-CLG bacteria; 5. ultrasonic precipitation of pET32a bacteria.
  • cDNA clones are used to transfect cancer cells.
  • sequencing proves that they are new genes, and further obtains full-length cDNA clones.
  • the DNA transfection test proves that the protein with tumor suppressing function of the present invention has the effect of inhibiting the formation of clones on cancer cells (liver cancer cells), and the inhibition rate is 50%.
  • a new gene with high homology to the cell cycle regulatory proteins of nematodes, Drosophila and yeast was isolated from the human placental cDNA library. It has 66% (557/837) identity to the cell cycle regulatory proteins of nematodes. Derived, has 41% (157/375) homology with the cell cycle regulatory protein (crooked neck, crn) of Drosophila, and 37 ° / with the cell cycle control protein of yeast. (228/599) Homology. According to bioinformatics, human CLG (crn-ike gene; original serial number is PP3898, GenBank accession number AF258567, registration date April 24, 2000) is a new human cell cycle-related gene.
  • CLG can inhibit the growth of human liver cancer cells SMMC-7721 in vitro; in situ apoptosis detection and DNA electrophoresis analysis of hepatocellular carcinoma S-cut C-7721 nude mice transplanted tumor tissues showed that CLG gene transfected with S-cut C-7721 cells can induce tumor cell apoptosis and inhibit tumor cell growth.
  • “cell cycle control related gene proteins”, “CLG protein”, and “PP3898 protein” may Used interchangeably, they all refer to polypeptides having the amino acid sequence (SEQ ID NO: 2) of a human cell cycle control-related gene protein. They include cell cycle control related gene proteins with or without starting methionine.
  • 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 separated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances coexisting in the natural state. of.
  • isolated CLG protein or polypeptide means that a CLG protein polypeptide is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify CLG proteins using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of CLG protein can be analyzed by amino acid sequence.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of the human CLG protein.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the natural human CLG protein of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of a polypeptide, such as (Polyethylene glycol), a polypeptide formed by fusion, or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify the polypeptide or a protein sequence). According to the teachings herein, these fragments, derivatives, and analogs are within the scope of those skilled in the art.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 3 or a degenerate variant.
  • degenerate variant refers in the present invention to a nucleic acid sequence that encodes a protein having SEQ ID NO: 2 but differs from the coding region sequence shown in SEQ ID NO: 3.
  • a polynucleotide encoding a mature polypeptide includes: a coding sequence that encodes only a mature polypeptide; a coding sequence that is a mature polypeptide
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and / or non-coding sequences.
  • the present invention also relates to a variant of the above polynucleotide, which encodes a polypeptide having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptide.
  • 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 hybridizing to the sequence described above with at least 50%, preferably at least 70% 'between the two sequences. More preferably a polynucleotide that is at least 80% identical.
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 “C; or ( 2) A denaturant is added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%, 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 fragment that hybridizes to the sequence described above.
  • a "nucleic acid fragment” contains at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding CLG proteins.
  • DNA sequence of the present invention can be obtained by several methods.
  • DNA is 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 nucleotide sequences, and 2) antibody screening of expression libraries to detect cloned DNA fragments with common structural characteristics .
  • the specific DNA fragment sequence encoding CLG protein can also be obtained by: 1) isolating double-stranded DNA sequence from genomic DNA; 2) chemically synthesizing DNA sequence to obtain double-stranded DNA of desired polypeptide.
  • genomic DNA isolation is the least commonly used.
  • direct chemical synthesis of the DNA sequence is often the method of choice. If the entire sequence of the desired amino acid is unclear, direct chemical synthesis of the DNA sequence is not possible, and the method chosen is the isolation of the cDNA sequence.
  • 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. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene).
  • cDNA libraries are also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, 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):
  • DNA-DNA or DNA-RNA hybridization (2) the occurrence or loss of the function of a marker gene; (3) determination of the level of CLG protein transcripts; (4) detection by immunological techniques or determination of biological activity Gene-expressed protein product.
  • 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 has a length of at least 15 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 2 kb, preferably within 1 kb.
  • the probe used herein is generally a DNA sequence chemically synthesized based on the DNA sequence information of the gene of the present invention.
  • the gene itself or a fragment of the present invention can of course be used as a probe.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of CLG protein gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method for amplifying DNA / RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the RACE method RACE- rapid cDNA end amplification method
  • the primers used for PCR can be based on the sequence information of the present invention disclosed herein. The information is appropriately selected and can be formed by a conventional method.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • nucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger t a). PNAS, 1977, 74: 5463-5467. Such nucleotide sequence determination can also be performed using a commercial sequencing kit or the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, a host cell genetically engineered using the vector or CLG protein coding sequence of the present invention, and a method for producing a polypeptide of the present invention by recombinant technology.
  • polynucleotide sequences of the present invention can be used to express or produce recombinant CLG protein polypeptides by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally there are the following steps:
  • the human CLG protein polynucleotide sequence can be inserted into a recombinant expression vector.
  • recombinant expression 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-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors (Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells.
  • any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes and translation control elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing human CLG protein-encoding DNA sequences and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • 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 vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein.
  • the host cell can be 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.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CH0, COS or Bowes melanoma cells.
  • Enhancers are cis-acting factors of DNA, 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 on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used.
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the obtained transformants can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional mediums. Culture is carried out 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.
  • the recombinant polypeptide in the above method may be coated intracellularly, extracellularly, or expressed on a cell membrane or secreted extracellularly. If necessary, the physical, chemical, and other properties can be used to separate and purify the recombinant protein by various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmosis, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with a protein precipitant (salting out method), centrifugation, osmosis, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis,
  • Recombinant human CLG proteins or polypeptides have many uses. These uses include (but are not limited to): direct use as a drug to treat diseases caused by hypofunction or loss of CLG protein, and to screen for antibodies, peptides, or other ligands that promote or fight CLG protein function. For example, antibodies can be used to activate or inhibit the function of human CLG proteins. Screening peptide libraries with expressed recombinant human CLG proteins can be used to find therapeutic peptides that can inhibit or stimulate the function of human CLG proteins.
  • the invention also provides methods for screening drugs to identify agents that increase (agonist) or suppress (antagonist) human CLG proteins.
  • Agonists enhance human CLG proteins 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 CLG proteins can be cultured with labeled human CLG proteins in the presence of drugs. The ability of the drug to increase or block this interaction is then measured.
  • Antagonists of human CLG proteins include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of the human CLG protein can bind to the human CLG protein and eliminate its function, or inhibit the production of the human CLG protein, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions. Antagonists of human CLG proteins are useful in therapeutic applications.
  • CLG proteins 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 the CLG protein and its receptor. In the same manner as described above for the screening of compounds, it is possible to screen for receptor deletions and analogs that act as antagonists.
  • the polypeptide of the present invention can be directly used for treating diseases, for example, various malignant tumors, abnormal cell proliferation, and the like.
  • the polypeptides of the present invention, and fragments, derivatives, analogs or their cells can be used as antigens to produce antibodies.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • Polyclonal antibodies can be obtained by directly injecting the polypeptide Material method. Techniques for preparing monoclonal antibodies include hybridoma technology, triple tumor technology, human B-cell hybridoma technology, and EBV-hybridoma technology.
  • the polypeptides and antagonists 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 that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that produce, use, or sell them.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • the CLG protein is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of CLG protein 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 polynucleotide of the human CLG protein can also be used for a variety of therapeutic purposes.
  • Gene therapy techniques can be used to treat abnormal cell proliferation, development, or metabolism caused by non-expressed CLG protein expression or abnormal / inactive CLG protein expression.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated CLG proteins to inhibit endogenous CLG protein activity.
  • a mutated CLG protein may be a shortened CLG protein lacking a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of CLG protein.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer CLG protein genes into cells. Methods for constructing a recombinant viral vector carrying the CLG protein gene can be found in existing literature (Sambr 00 k, et al.). In addition, the recombinant human CLG protein gene can be packaged into liposomes and transferred into cells.
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human CLG protein mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, 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 ribonucleosides are connected by phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell via a vector (such as a virus, phage, or plasmid) in vitro Then, the cells are transplanted into the body.
  • a vector such as a virus, phage, or plasmid
  • the invention also provides antibodies against human CLG protein 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. Anti-human CLG protein antibodies can be used in immunohistochemical techniques to detect human CLG protein in biopsy specimens.
  • Monoclonal antibodies that bind to human CLG proteins 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.
  • the antibodies in the present invention can be used to treat or prevent diseases related to human CLG protein. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human CLG protein.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human CLG protein 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 the 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 CLG protein-positive cells.
  • Polyclonal antibodies can be produced by immunizing animals such as rabbits, mice, and rats with human CLG proteins or peptides.
  • immunizing animals such as rabbits, mice, and rats with human CLG proteins or peptides.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.
  • Human CLG protein monoclonal antibodies can be produced using hybridoma technology (Kohler and Ilstein. Nature, 1975, 256: 495-497). Chimeric antibodies that bind human constant regions to non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). The existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against human CLG protein.
  • Polypeptide molecules capable of binding to human CLG protein 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 CLG protein molecule must be labeled.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human CLG protein.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human CLG protein detected in the test can be used to explain the importance of human CLG protein in various diseases and to diagnose diseases in which CLG protein functions.
  • CLG protein polynucleotides are useful in the diagnosis and treatment of CLG protein-related diseases.
  • diagnosis the polynucleotide of CLG protein can be used to detect the expression of CLG protein or the abnormal expression of CLG protein in a disease state.
  • the CLG protein DNA sequence can be used to hybridize biopsy specimens to determine abnormal CLG protein expression.
  • Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These technical methods are publicly available and mature technologies, 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 known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • CLG protein-specific primers for RNA-polymerase chain reaction (RT-PCR) amplification in vitro can also detect CLG protein transcripts.
  • CLG protein mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type CLG protein 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, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared from the cDNA, and the sequence can be mapped on the chromosome. 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.
  • a similar oligonucleotide primer can be similarly used to achieve sublocalization using a set of fragments from a specific chromosome or a large number of genomic clones.
  • Other similar strategies that can be used for staining 'localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal mapping in a single step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the gene map data. These data can be found in, for example, V. Mckusick, Mendel ian Inheri tance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution Capacity and each 20kb corresponds to a gene).
  • the full-length nucleotide sequence of the CLG protein of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method.
  • primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or cDNA prepared according to conventional methods known to those skilled in the art
  • the library is used as a template and the relevant sequences are amplified. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then stitch the amplified fragments together in the correct order.
  • the relevant sequence can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • relevant methods can also be synthesized by artificial synthesis, especially when the fragment length is short.
  • long sequences can be obtained by first synthesizing multiple small fragments and then concatenating them.
  • the DNA sequence encoding the protein (or a fragment, or a derivative thereof) of the present invention can be completely synthesized by chemical synthesis. This DNA sequence can then be introduced into various DNA molecules (such as vectors) and cells in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the CLG protein of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and / or lower side effects when administered to humans compared to homologous proteins derived from other species ( (Eg, less or not immunogenic in humans).
  • the present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.
  • the experimental methods without specific conditions in the examples are generally according to the conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions. Suggested conditions.
  • Example 1 Obtaining a cDNA Gene Fragment and Inhibiting the Formation of Cancer Cell Clones
  • PP3898 comes from the construction of a human placental cDNA library using conventional methods. Take 3, 6, and 10-month-old placental tissues, use Trizol reagent (GIBC0 BRL company) to extract total RNA according to the manufacturer's instructions, and use mRNA to purify the reagent Cassette (Pharmacia) for mRNA extraction. A pCMV-script TMXR cDNA library construction kit (Seratagene) was used to construct a cDNA library of the above-mentioned I RNA. The reverse transcriptase was changed to MMLV-RT- Superscript II (GIBC0 BRL), and the reverse transcription reaction was performed at 42 ° C.
  • XL 10-Gold receptor cells were transformed to obtain a cDNA library with a lxlO 6 cfu / g cDNA titer.
  • cDNA clones were randomly selected, and thereafter, high-abundance cDNA clones and cDNA clones that had been shown to inhibit the growth of cancer cells were used as probes to hybridize and screen cDNA libraries, and weakly positive and negative clones were selected.
  • Qiagen 96-well plate plasmid extraction kit was used to extract plasmid DNA according to the manufacturer's instructions. Plasmid DNA and empty vector were transfected into hepatocellular carcinoma cell line 7721 at the same time.
  • PP3898 ie CLG 2 0 0 33 34 38
  • the dideoxy termination method was used for cDNA clones, and the nucleotide sequence of one end of the clone was nearly 500bp on an ABI377 DNA automatic sequencer. After analysis, it was determined to be a new gene clone, and then completely sequenced. It was found that the CLG fragment was a non-full-length sequence, and the full-length cDNA clone was obtained by the RACE method.
  • Example 2 RACE method to obtain full-length cDNA clone and RT-PCR method to obtain CLC gene
  • the SMART RACE cDNA amplification kit (Cat. No. K1811-1) from Clontech was used to design gene specific primers (shown in Table 2 below). Obtain full-length clones.
  • the human placental tissue mRNA was used as the starting material, and the cDNA was obtained according to the instructions of Clontech's SMART RACE cDNA Amplification Kit (Catftl811-1). Then the first round of PCR was performed with UPM primers and gene-specific pp3898-B primers, and then NUP Primers and gene-specific pp3898-NB primers were subjected to a second round of PCR to obtain gene fragments.
  • reaction conditions are as follows: 94 ° C for 1 minute, one cycle; 94 ° C for 30 seconds, 72 ° C for 4 minutes, 5 cycles; 94 ° C 30 seconds, 70 ° C 4 minutes, 5 cycles; 94 ° C 20 seconds, 65 ° C 30 seconds, 68 ° C 4 minutes, 27 cycles.
  • a SMART RACE reaction was performed to obtain a 5 'extension of the CLG gene, which was recombined to obtain a cDNA clone of the full-length CLG gene, as shown in SEQ ID NO: 1.
  • GAGCCCGTGC AGAACCGCGT GTACAAGTCA CTGAAGGTCT GGTCCATGCT CGCCGACCTG 1500
  • the homology analysis of the amino acid sequence of CLG was found to be highly homologous with the cell cycle regulatory proteins of Drosophila, nematodes and yeast: 41% with the cell cycle regulatory proteins (crooked neck, crn) of Drosophila ( 157/375). It has 66% (557/837) homology with the cell cycle regulatory protein of nematodes and 37% (228/599) homology with the cell cycle control protein of yeast.
  • members of the TPR gene family in yeast include the negative regulator SSN6 of the sucrose-inducible gene, the negative regulator SKI3 of yeast killer toxin and the mitochondrial membrane protein MAS70 related to protein input.
  • the CLG gene is the same as these Drosophila and yeast genes. This conserved motif is repeated in series in straight lines in the protein sequence of CLG . It appears repeatedly and has commonality.
  • CLG was found in human heart (H), brain (B), placenta (P), lung (Lu), liver (Li), muscle (SM), kidney (K), and pancreas (Pa) tissues. It is widely expressed, and the expression level is relatively uniform, in which the transcript size in human brain, placenta, lung, liver, kidney and pancreas is about 2. 6kb, which is the same as the full-length cDNA size of CLG obtained in Example 2. Ok, a transcript of about 3. Okb appears in the heart and muscle tissue.
  • Example 6 Inhibitory effect of CLG on colony formation of human liver cancer cells
  • the CLG gene containing the complete coding region obtained by RT-PCR was cloned into the pT-Adv vector (Clontech) using AdvanTAge TM PCR Cloning reagent (Clontech, Cat #: K1901- 1), and then digested with EcoR I
  • the CLG gene fragment containing the complete coding region was recovered and subcloned into the eukaryotic cell expression vector pCMV-
  • the plasmid DNA was extracted using the Qiagen plasmid extraction kit for transfection.
  • human liver cancer cells SMMC-7721 were transfected with CLG gene, and then the transformed cells and control cells not transfected with CLG gene were expanded and cultured.
  • Nude mice were subcutaneously inoculated to observe the effect of CLG gene on tumorigenesis. The experiment was divided into a CLG transfection group and a control group. Each nude mouse was inoculated with 2 ⁇ 10 6 cells. The observation period was 6 weeks. Table 3 shows the tumor formation results.
  • CLG-transfected S-band C-7721 cells were inoculated with tumor tissues formed in nude mice and used as paraffin sections.
  • In situ detection of apoptosis by Tunel method (Roche) blue-violet positive apoptotic cells were seen, among which necrosis There were more positive apoptotic cells in the focus (shown in Figure 3A), and scattered apoptotic cells were seen in the non-necrotic area (shown in Figure 3B).
  • primer 3 5 'TCGGAATTCGTGGTGATGGCGCGACTT 3', 5 'end contains EcoR I site
  • primer 4 5' TCTAAGCTTTCAGTCTTCCTTCAGGCT 3 ', 5' end contains Hind III site
  • the PCR reaction conditions were: 94 ° C, denaturation for 5 min; then denaturation at 94 ° C for 1 min, annealing at 55 ° C for 1 min, extension at 72 ° C for 2 min, and performing 40 cycles; finally, extension at 72 ° C for 10 min. After the reaction product is detected by electrophoresis, it is recovered by enzyme digestion.
  • the fragment of the CLG coding region recovered by PCR amplification and digestion was inserted into the EcoR I-Hind III site of the vector pET32a (Novagen) to obtain a pET32a-CLG recombinant plasmid.

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Abstract

L'invention concerne une nouvelle protéine humaine anti-tumorale et un polynucléotide codant pour la protéine ainsi qu'un procédé d'obtention d'un polypeptide par technique recombinante. L'invention concerne également des applications de ce polypeptide dans le traitement de différentes maladies, notamment les tumeurs. L'invention concerne en outre un antagoniste dirigé contre le polypeptide et son utilisation thérapeutique. Fait aussi l'objet de cette invention un polynucléotide codant pour la nouvelle protéine humaine anti-tumorale.
PCT/CN2001/000121 2000-02-17 2001-02-12 Nouvelle proteine humaine associee a la regulation du cycle cellulaire et sa sequence de codage WO2001060855A1 (fr)

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WO2003027279A1 (fr) * 2001-09-25 2003-04-03 Sony Corporation Inhibiteur de l'histone acetylase de p300
WO2004031388A1 (fr) * 2002-07-25 2004-04-15 Sony Corporation Facteur participant a la regulation de la transcription

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CN101995472B (zh) * 2009-08-21 2013-10-09 中国科学院上海生命科学研究院 一种细胞周期检查点调控蛋白用作检测肝细胞癌的蛋白质分子标记的应用
CN117624311B (zh) * 2023-11-29 2024-05-28 广州译码基因科技有限公司 可提高aav病毒神经靶向性的衣壳蛋白突变体及其应用

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RNA, vol. 5, no. 8, 1999, pages 1042 - 1054 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003027279A1 (fr) * 2001-09-25 2003-04-03 Sony Corporation Inhibiteur de l'histone acetylase de p300
US7834169B2 (en) 2001-09-25 2010-11-16 Sony Corporation P300 histone acetylase inhibitor
US8309700B2 (en) 2001-09-25 2012-11-13 Sony Corporation Inhibitor of histone acetyltransferase, especially p300
WO2004031388A1 (fr) * 2002-07-25 2004-04-15 Sony Corporation Facteur participant a la regulation de la transcription
JPWO2004031388A1 (ja) * 2002-07-25 2006-02-02 ソニー株式会社 転写調節に関与する因子
JP4508872B2 (ja) * 2002-07-25 2010-07-21 ソニー株式会社 遺伝子転写調節剤
US8889408B2 (en) 2002-07-25 2014-11-18 Sony Corporation Factor taking part in transcription control

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