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WO2001038522A1 - Nouveau polypeptide, histone humaine h2a.21, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, histone humaine h2a.21, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001038522A1
WO2001038522A1 PCT/CN2000/000440 CN0000440W WO0138522A1 WO 2001038522 A1 WO2001038522 A1 WO 2001038522A1 CN 0000440 W CN0000440 W CN 0000440W WO 0138522 A1 WO0138522 A1 WO 0138522A1
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Prior art keywords
polypeptide
polynucleotide
human histone
sequence
seq
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PCT/CN2000/000440
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Bioroad Gene Development Ltd. Shanghai
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Priority to AU15086/01A priority Critical patent/AU1508601A/en
Publication of WO2001038522A1 publication Critical patent/WO2001038522A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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 polypeptide, a human histone H2A. 21, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing such polynucleotides and polypeptides. Background technique
  • Nucleosomes are the basic structural units that make up chromatin, making DNA, RNA, and protein tissues in chromatin a dense structural form. Each nucleosome unit contains about 200bp of DNA and a histone octamer and a molecule of histone Hl. There are five major histones on the chromosomes of general eukaryotes, named H1, H2A, H2B, H3, and H4. They are the same in different tissues of the same organism, and they are similar in different eukaryotes. Of the five histones, in addition to HI, the other four have a tendency to interact to form aggregates.
  • H2A family has four subtypes that differ in length and gene sequence.
  • Their synthesis is always consistent with DNA replication.
  • Nucleosomes play important roles in maintaining the shape and structure of chromosomes and regulating gene expression in eukaryotes.
  • the present invention has 98% homology with human histone H2A. 2 at the protein level. Therefore, the gene of the present invention is considered to be a gene encoding a protein similar to histone H2A. 2 and has similar biological functions.
  • the present invention is named human histone H2A. 21. As mentioned above, the human histone H2A.
  • the human histone H2A. 21 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 involved in these processes.
  • the human histone H2A. 21 protein in particular, identifies the amino acid sequence of this protein. Isolation of the new human histone H2A. 21 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human histone H2A.21.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human histone H2A.21.
  • Another object of the present invention is to provide a method for producing human histone H2A.21.
  • Another object of the present invention is to provide an antibody against the polypeptide-human histone H2A. 21 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human histone H2A.21.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to human histone H2A. 21 abnormalities.
  • 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 No. 2 or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is
  • ID NO: 2 polypeptide of amino acid sequence.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 114-698 in SEQ ID NO: 1; and (b) a sequence having 1-948 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector containing a polynucleotide of the present invention, particularly an expression vector; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said 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 histone H2A. 21 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 in vitro detection of a disease or susceptibility to disease associated with abnormal expression of a human histone H2A. 2 1 protein, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting The amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to 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 in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human histone H2A.21.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and can also refer to genomic or synthetic DNA or RNA, which can be single-stranded or double-stranded, representing the sense strand or Antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and a fragment or part thereof.
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acid 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 substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of 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.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunological activity refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind to specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human histone H2A. 21, 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 histone H2A.21.
  • Antagonist refers to a molecule that, when combined with human histone H2A. 21, can block or regulate the biological or immunological activity of human histone H2A. 21.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind human histone H2A.21.
  • Regulation refers to a change in the function of human histone H2A. 21, including an increase or decrease in protein activity, Changes in binding properties and any other biological, functional or immune properties of human histone H2A.21.
  • substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human histone H2A.21 using standard protein purification techniques.
  • the substantially pure human histone H2A.21 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human histone H2A.21 polypeptide can be analyzed by amino acid sequence analysis.
  • Complementary refers to the natural binding of a polynucleotide by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T- G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules can 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 the hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences.
  • the percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.).
  • the MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244).
  • the Clus ter method compares each group of sequences by checking the distance between all pairs. The clusters are arranged. The clusters are then allocated in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotim Hein (Hein J., (1990) Methods in emzumology 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamic acid Aminoamides; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be a substitution of a hydrogen atom with a fluorenyl group, an acyl group or an 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 the epitope of human histone H2A. 21.
  • 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 occurs naturally).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, 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 vector, or it may be such a polynucleotide or peptide that is part of a composition. Since the carrier or composition is not part of its natural environment, they are still separate.
  • 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 histone H2A. 21 means human histone H2A. 21 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 histone H2A. 21 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of human histone H2A. 21 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human histone H2A. 21, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human histone H2A.21. As used in the present invention, the terms “fragment”, “derivative” and “analog” refer to a polypeptide that substantially retains the same biological function or activity of the human histone H2A. 21 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: U) a type in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted
  • the amino acid may or may not be encoded by the genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III) such a Species, 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 the additional amino acid sequence is fused into the mature polypeptide (such as Leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes 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 full-length polynucleotide sequence of 948 bases, and its open reading frame (114-698) encodes 194 amino acids. According to the homologous comparison of amino acid sequences, it was found that this peptide has 98% homology with h s t oneH2A. 1, and it can be inferred that the human histone H2A. 21 has a similar structure and function to h s tone H2A. 1.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the sequence of the coding region encoding a mature polypeptide can be as shown in SEQ ID NO: 1
  • the coding region sequence shown is the same or a degenerate variant.
  • a "degenerate variant" refers to a protein or polypeptide having SEQ ID NO: 2 in the present invention, but is identical to SEQ ID NO: Nucleic acid sequences having different coding region sequences shown in 1.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may 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 polynucleoside, which may be a Substitutions, deletions or insertions of one or more nucleotides without substantially altering the function of the polypeptide it encodes.
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • "strict conditions” means: (1) 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 50 ° /.
  • 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 histone H2A.21.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • polynucleotide sequence encoding the human histone H2A.21 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) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DM of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for mRNA extraction, and kits are also commercially available (Qiagene). Construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, even very small expression products can be cloned.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human histone H2A.21 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 by PCR is preferably used to obtain the gene of the present invention.
  • the RACE method RACE- rapid amplification of cDNA ends
  • the primers for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.
  • the polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequencing can also be performed using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human histone H2A.21 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .
  • a polynucleotide sequence encoding human histone H2A.21 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 an origin of replication, a promoter, marker genes, and translational regulatory elements.
  • DM sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site 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. 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.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human histone H2A. 21 or a recombinant vector containing the polynucleotide can be transformed or introduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be harvested after exponential growth phase, with (: Treatment 1 2, steps well known in the art with alternative is MgC l 2
  • transformation can also be performed by electroporation.
  • the host is a eukaryote
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipids. Body packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human histone H2A. 21 (Scence, 1 984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional media. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • 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. 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 comparison diagram of the amino acid sequence homology of the human histone H2A.21 and histoneH2A, 1 of the present invention.
  • the upper sequence is human histone H2A.21 and the lower sequence is histoneH2A.1.
  • the same amino acid is represented by a single-character amino acid between the two sequences, and similar amino acids are represented by "+".
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human histone H2A.21. 21kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (product of Qiegene). 2ug poly (A) mRNA was reverse transcribed to form cDNA.
  • Directional insertion of cDNA fragments into pBSK (+) vector (Clomech using Smart cDNA Cloning Kit (purchased from Clontech) The company cloned DH5 cc at multiple cloning sites, and the bacteria formed a cDNA library.
  • Dye terminate cycle reaction sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 1003D09 was new DNA.
  • a series of primers were synthesized to perform bidirectional determination of the inserted CDM fragments contained in this clone.
  • 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, PCR was performed using the following primers:
  • Primerl 5'- GGCCGCGGTCTAGCCTACCTCTG -3 '(SEQ ID NO: 3)
  • Primer2 5'- GATCATAAATACTTTTAATATCTG -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 mmol / L KC1, 10 mmol / L Tris-Cl, ( ⁇ 8.5 ⁇ ), 1.5 mmol / LMgCl 2 , 200 mol / LdNTP, lOpmol primer, 1U Taq in a reaction volume of 50 ⁇ 1 DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -actin was set as a positive control and template blank was used as a negative control.
  • the amplified product was purified using a QIAGEN kit, and ligated to a pCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-948b P shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human histone H2A.21 gene expression:
  • RNA extraction in one step involves acid guanidinium thiocyanate phenol-chloroform extraction. 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M acetic acid Sodium (pH 4.0) was used to homogenize the tissue, 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) were added, and the mixture was centrifuged. The aqueous phase layer was aspirated and isopropyl alcohol (0.8 Volume) and centrifuge the mixture to obtain an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA in 20 mM 3- (N-morpholino) propanesulfonic acid ( ⁇ 7 ⁇ 0) -5 mM Sodium Acetate-1.2 mM EDTA-2.2M formaldehyde 1.2. / »Agarose gel for electrophoresis. Then transfer to a nitrocellulose membrane.
  • Prepare labeled DNA by random primer method using ⁇ - 32 P dATP Probes.
  • the DNA probes used were the PCR-encoded human histone H2A.21 coding region sequence (U4bp to 698b P ) as shown in Figure 1.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was used.
  • Primer4 5'- CATGGATCCTCAGTCCCCAGGGTCTCGGTTCCCG -3 '(Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • the Nde I and BamH I restriction sites correspond to the expression vector plasmid pET- 28 b (+) (Novagen, Cat. No. 69865.3) a selective endonuclease site.
  • the pBS-1003D09 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ 1 contains 10 pg of pBS-1003D09 plasmid, primer Primer-3 and?
  • the positive clones with correct sequence were selected and recombined by the calcium chloride method.
  • the plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen).
  • E. coli BL21 DE3 plySs (product of Novagen).
  • the host strain BL21 pET-1003D09 was cultured at 37 ° C.
  • IPTG IPTG
  • a final concentration of 1 mmol / L add IPTG to a final concentration of 1 mmol / L, and continue to cultivate for 5 hours. Centrifuge the bacteria to collect the bacteria, sonicate the bacteria, and centrifuge to collect the supernatant.
  • Bind Quick Cartridge (product of Novagen) was chromatographed to obtain Of human protein histone H2A.21. By SDS- PAGE electrophoresis, the obtained at 21kDa To a single band ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2.
  • a peptide synthesizer (product of PE company) was used to synthesize the following human histone H2A. 21-specific peptides:
  • polypeptide of the present invention as well as its antagonists, agonists and inhibitors, can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and immune diseases.
  • Nucleosomes play important roles in maintaining the shape and structure of chromosomes and regulating gene expression in eukaryotes. Deletion of histones or mutations in histone genes will affect the function of nucleosomes, leading to chromosomal replication and abnormal gene expression, which will cause a series of serious diseases, such as developmental disorders, metabolic diseases, immune diseases and even cancer. .
  • the inventor's histone H2A. 21 can be used to treat and prevent developmental disorders caused by abnormal chromosomal replication, including but not limited to: Down's syndrome, Trisomy 18, 1 3 Trisomy syndrome, 4p trisomy syndrome, 9p trisomy syndrome, etc.
  • the polypeptide or fragment thereof of the present invention can also be used to treat or prevent diseases caused by metabolic disorders, including but not limited to: phenylketonuria, albinism , Trimethylamineuria, hypersarcosinemia, etc .; the polypeptides or fragments thereof of the present invention can also be used to treat or prevent various immunodeficiency disorders, including but not limited to: HIV, rheumatoid arthritis, chronic activity Hepatitis, systemic lupus erythematosus, scleroderma, immune thrombocytopenic purpura, autoimmune interstitial nephritis, autoimmune heart disease, etc.
  • diseases caused by metabolic disorders including but not limited to: phenylketonuria, albinism , Trimethylamineuria, hypersarcosinemia, etc .
  • the polypeptides or fragments thereof of the present invention can also be used to treat or prevent various immunodeficiency disorders, including but not limited to: HIV, r
  • the polypeptides or fragments thereof of the present invention can also be used to treat or prevent various tumors, including but not limited to: nasal and sinus tumors, nasopharyngeal carcinoma, laryngeal cancer, tracheal tumor, lung cancer, pleural mesothelioma, salivary gland tumor, esophageal cancer , Esophageal leiomyosarcoma, primary esophageal small cell carcinoma, gastric cancer, gastric malignant lymphoma, gastric carcinoid, colorectal cancer, colon cancer, intestinal malignant lymphoma, primary liver cancer, hepatoblastoma, primary gallbladder Cancer, pancreatic cancer, acute white blood Disease, chronic myelogenous leukemia, chronic lymphocytic leukemia, malignant lymphoma (such as lymphatic reticulum, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), malignant his
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human histone H2A.21.
  • Agonists enhance human histone H2A. 21 stimulates biological functions such as cell proliferation, while antagonists block and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human histone H2A. 21 can be cultured with labeled human histone H2A. 21 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human histone H2A. 21 include screened antibodies, compounds, receptor deletions and analogs. Antagonists of human histone H2A. 21 can bind to human histone H2A. 21 and eliminate its function, or inhibit the production of the peptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human histone H2A. 21 can be added to a bioanalytical assay to determine whether the compound is antagonistic by measuring the effect of the compound on the interaction between human histone H2A. 21 and its receptor. Agent. 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 histone H2A. 21 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, the human histone H2A. 21 molecule should generally 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 an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human histone H2A. 21 epitope.
  • These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced using human histone H2A. 21 direct injection in immunized animals (such as rabbits, mice, rats Rat, etc.), a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies against human histone H2A. 21 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma Technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morr is on et al, PNAS, 1985, 81: 6851). 0
  • Existing techniques for producing single-chain antibodies can also be used to produce single chain antibodies against human histone H2A.21.
  • Antibodies against human histone H2A. 21 can be used in immunohistochemistry to detect human histone H2A. 21 in biopsy specimens.
  • Monoclonal antibodies that bind to human histone H2A. 21 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human histone H2A. 21 High affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human histone H2A. 21 positive cells .
  • the antibodies of the present invention can be used to treat or prevent human histone H2A. 21-related diseases. Administration of an appropriate amount of antibody can stimulate or block the production or activity of human histone H2A.21.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human histone H2A. 21 levels. These tests are well known in the art and include FI SH assays and radioimmunoassays. The level of human histone H2A. 21 detected in the test can be used to explain the importance of human histone H2A. 21 in various diseases and to diagnose diseases where human histone H2A. 21 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding human histone H2A. 21 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 histone H2A.21. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human histone H2A. 21 to inhibit endogenous human histone H2A. 21 activity.
  • a mutated human histone H2A. 21 may be a shortened human histone H2A. 21 lacking a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human histone H2A.21.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human histone H2A. 2 1 into a cell.
  • Construct Methods for carrying recombinant viral vectors carrying a polynucleotide encoding human histone H2A. 21 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human histone H2A. 21 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 histone H2A. 21 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 phosphate amide 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 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 phosphorothioate or peptide bond instead of the phosphodiester bond should be used for the ribonucleoside linkage.
  • the polynucleotide encoding human histone H2A. 21 can be used for the diagnosis of diseases related to human histone H2A. 21.
  • a polynucleotide encoding human histone H2A. 21 can be used to detect the expression of human histone H2A. 21 or the abnormal expression of human histone H2A. 21 in a disease state.
  • the DNA sequence encoding human histone H2A. 21 can be used to hybridize biopsy specimens to determine the expression of human histone H2A. 21.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are available commercially.
  • a part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in a tissue.
  • 21 specific primers can also be used to detect human histone H2A.
  • 21 transcripts by RNA-polymerase chain reaction (RT-PCR) in vitro amplification.
  • Detection of mutations in the human histone H2A. 21 gene can also be used to diagnose human histone H2A. 21-related diseases.
  • Human histone H2A. 21 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human histone H2A. 21 DNA sequences. Mutations can be detected using existing techniques such as Sou thern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins, so Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • Only a few chromosome markers based on actual sequence data (repeating polymorphisms) are now available for labeling chromosomes Location.
  • an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells containing human genes corresponding to the primers will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, sublocalization can be achieved using a set of fragments from a specific chromosome or a large number of genomic clones.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones to metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all 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 cDM sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition contains 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.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders authorize them to be administered to humans by government agencies that manufacture, use, or sell them.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human histone H2A. 21 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of human histone H2A. 21 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.
  • Ala Glu lie Leu Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys

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Abstract

L'invention concerne un nouveau polypeptide, une histone humaine H2A.21, 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 l'histone humaine H2A.21.
PCT/CN2000/000440 1999-11-23 2000-11-20 Nouveau polypeptide, histone humaine h2a.21, et polynucleotide codant pour ce polypeptide WO2001038522A1 (fr)

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CN99124083A CN1296974A (zh) 1999-11-23 1999-11-23 一种新的多肽——人组蛋白h2a.21和编码这种多肽的多核苷酸
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Cited By (5)

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EP1572069A2 (fr) * 2001-08-29 2005-09-14 Yissum Research Development Company of the Hebrew University of Jerusalem Facteurs de protection contre des inflammations, des brulures et des stimuli nocifs
EP1593385A1 (fr) * 2004-05-07 2005-11-09 SYMBIOTEC Gesellschaft zur Forschung und Entwicklung auf dem Gebiet der Biotechnologie mbH Utilisation des histones pour le diagnostic et/ou la thérapie préventive des cellules vivantes infectées par des virus et une biopuce pour l'exécution du diagnostic.
US7238656B2 (en) 2001-08-29 2007-07-03 Yissum Research Development Company Of The Hebrew University Of Jerusalem Protective factors against inflammation, burns and noxious stimuli
US7528227B2 (en) 2004-03-23 2009-05-05 Yissum Research Development Company Of The Hebrew University Of Jerusalem Histone H2A peptide derivatives and uses thereof
US7605132B2 (en) 2001-08-29 2009-10-20 Yissum Research Development Company Of The Hebrew University Of Jerusalem Protective factors against inflammation, burns and noxious stimuli

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DE3737274A1 (de) * 1987-11-03 1989-06-01 Rusch Volker Verwendung von reinem h2a- und/oder h2b-histon als wirksubstanz zur herstellung von arzneimitteln
EP0392315A1 (fr) * 1984-01-12 1990-10-17 SYMBIOTEC Gesellschaft zur Forschung und Entwicklung auf dem Gebiet der Biotechnologie GmbH Histone H1 pure utilisable dans des procédés thérapeutiques
US5330971A (en) * 1989-06-09 1994-07-19 Gropep Pty. Ltd. Growth hormone fusion proteins, methods of production, and methods of treatment
US5578571A (en) * 1990-01-04 1996-11-26 Symbiotec Gesellschaft Zur Forschung Und Entwicklung Auf Dem Gebiet Der Biotechnologie Mbh Cytostatic or cytotoxic combination of active substances for use in therapeutic procedures
US5714462A (en) * 1992-05-07 1998-02-03 Ml Laboratories Antiviral agent comprising CD4 and H2 histone

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EP0392315A1 (fr) * 1984-01-12 1990-10-17 SYMBIOTEC Gesellschaft zur Forschung und Entwicklung auf dem Gebiet der Biotechnologie GmbH Histone H1 pure utilisable dans des procédés thérapeutiques
DE3737274A1 (de) * 1987-11-03 1989-06-01 Rusch Volker Verwendung von reinem h2a- und/oder h2b-histon als wirksubstanz zur herstellung von arzneimitteln
US5330971A (en) * 1989-06-09 1994-07-19 Gropep Pty. Ltd. Growth hormone fusion proteins, methods of production, and methods of treatment
US5578571A (en) * 1990-01-04 1996-11-26 Symbiotec Gesellschaft Zur Forschung Und Entwicklung Auf Dem Gebiet Der Biotechnologie Mbh Cytostatic or cytotoxic combination of active substances for use in therapeutic procedures
US5714462A (en) * 1992-05-07 1998-02-03 Ml Laboratories Antiviral agent comprising CD4 and H2 histone

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1572069A2 (fr) * 2001-08-29 2005-09-14 Yissum Research Development Company of the Hebrew University of Jerusalem Facteurs de protection contre des inflammations, des brulures et des stimuli nocifs
WO2003017920A3 (fr) * 2001-08-29 2006-08-10 Yissum Res Dev Co Facteurs de protection contre des inflammations, des brulures et des stimuli nocifs
US7238656B2 (en) 2001-08-29 2007-07-03 Yissum Research Development Company Of The Hebrew University Of Jerusalem Protective factors against inflammation, burns and noxious stimuli
EP1572069A4 (fr) * 2001-08-29 2009-06-24 Yissum Res Dev Co Facteurs de protection contre des inflammations, des brulures et des stimuli nocifs
US7605132B2 (en) 2001-08-29 2009-10-20 Yissum Research Development Company Of The Hebrew University Of Jerusalem Protective factors against inflammation, burns and noxious stimuli
US7528227B2 (en) 2004-03-23 2009-05-05 Yissum Research Development Company Of The Hebrew University Of Jerusalem Histone H2A peptide derivatives and uses thereof
EP1593385A1 (fr) * 2004-05-07 2005-11-09 SYMBIOTEC Gesellschaft zur Forschung und Entwicklung auf dem Gebiet der Biotechnologie mbH Utilisation des histones pour le diagnostic et/ou la thérapie préventive des cellules vivantes infectées par des virus et une biopuce pour l'exécution du diagnostic.
WO2005112975A1 (fr) * 2004-05-07 2005-12-01 Symbiotec Gesellschaft Zur Erforschung Und Entwicklung Auf Dem Gebiete Der Biotechnology Mbh Utilisation d'histones pour le diagnostic precoce et/ou la therapie preventive de cellules vivantes infectees par un virus et une biopuce pour la mise en oeuvre du diagnostic
US9415089B2 (en) 2004-05-07 2016-08-16 Symbiotic Genellschaft zur Erforschung auf dem Geibeit der Biotechnologie, MBH Methods of treating virally-infected living cells with histones

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