WO2001027148A1 - Nouveau polypeptide, facteur homo 56 riche en glutamine(q), et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, facteur homo 56 riche en glutamine(q), et polynucleotide codant pour ce polypeptide Download PDF

Info

Publication number
WO2001027148A1
WO2001027148A1 PCT/CN2000/000310 CN0000310W WO0127148A1 WO 2001027148 A1 WO2001027148 A1 WO 2001027148A1 CN 0000310 W CN0000310 W CN 0000310W WO 0127148 A1 WO0127148 A1 WO 0127148A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
enrichment factor
human glutamine
seq
Prior art date
Application number
PCT/CN2000/000310
Other languages
English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
Original Assignee
Shanghai Bio Door Gene Technology Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Bio Door Gene Technology Ltd. filed Critical Shanghai Bio Door Gene Technology Ltd.
Priority to AU77683/00A priority Critical patent/AU7768300A/en
Publication of WO2001027148A1 publication Critical patent/WO2001027148A1/fr

Links

Classifications

    • 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 novel polypeptide-human glutamine enrichment factor 56, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing the polynucleotide and polypeptide.
  • Eukaryotic transcription factors are a class of proteins discovered in the past ten years that regulate the transcription of eukaryotic genes. They are involved in organisms to determine in which tissues and developmental stages genes begin to transcribe. If a gene encoding such a protein is mutated, not only the gene itself cannot be normally expressed, but also many genes regulated by it cannot be normally transcribed and expressed.
  • the regulation of transcription factors on gene expression is mainly accomplished through the binding of transcription factors to specific DNA sequences, the interaction between transcription factors, and the interaction of transcription factors with conventional transcriptional mechanisms. Studies on a variety of mammalian transcription factors have found that each transcription factor contains DNA binding domains of varying lengths composed of about 80-100 amino acid residues.
  • the DNA binding domain has four more fancy structures: ⁇ -helix-turn- ⁇ -helix; Cys-His zinc finger; Cys-Cys zinc finger; Leu-zipper. Fancy structure is important for maintaining the structure of the entire domain and the normal biological functions of transcription factors.
  • Hepatocyte nuclear factor 3 / fork head (HNF / fkh) protein family is ubiquitous in organisms. In recent years, many members of this protein family have been cloned from yeast to human. Further research found that members of this family all contained a characteristic domain consisting of 84-105 amino acid residues, an HNF / fkh domain of varying length. HNF / fkh protein family members can be divided into several different subclasses according to the original sequence of their domains. The family members of different subclasses have no obvious similarity except for the highly conserved DNA binding domain. The HNF / fkh domain is one of the DNA protein binding domains, and it is mainly involved in binding to specific DNA sequences in the body to regulate the expression of various genes.
  • HNF / fkh protein family have many important biological functions in the body, which are related to embryonic development, cell differentiation and proliferation, various organ formation, immune-deficient diseases such as AIDS, coronary heart disease, and various liver diseases. It has a certain relationship with the occurrence of various tumors and cancers (DaSliva L, Kirken RA et al., Gene 1998 22: 135-142; Frank S, Zoll B; DNA Cell Biol 1998 17: 679-88).
  • the HNF / fkh domain consists of 10 independent structural units. Its N-terminus has a hook-shaped structure followed by two hydrophilic spiral structures. The two helix structures are separated by a corner structure, the "helix-turn-alpha helix structure" in the fancy structure of the DNA binding domain. Among them, several conserved amino acid residues in the helical structure form two hydrophobic centers. These two hydrophobic regions are related to protein-protein interactions and the stability of the HNF / fkh domain. The only T23 residue in the second alpha helix is the recognition site for CK-2 kinase. The phosphorylation and dephosphorylation of this site may directly affect the DNA binding activity and HNF / fkh domain Formation of secondary structure.
  • this alpha helix has also been confirmed as a recognition helix, which is related to the recognition and binding of DNA. Its outer side is the main determinant of the specificity of protein-DNA binding; the fancy structure is followed by an aromatic residue and The third hydrophilic helix structure forms the central part of the domain; the C-terminus of the domain contains two variable-length loop structures, which are flanked by seven amino acid residues on both sides It consists of a short nucleotide chain. This short C-terminal nucleotide chain is the region where the domain interacts with DNA.
  • the hooked structure in this domain may be directly involved in protein-DNA interactions (Li C, et al. Proc. Natl. Acad Sci.
  • this domain plays an important role in the interaction of transcription factors with specific DNA sequences and regulating the expression of various genes.
  • the peptide is a typical HNF / fkh domain. Studies have shown that the polypeptide is involved in the development of lymphocytes and may also play a role in the development of leukemia (Li C, et al. Proc. Natl. Acad. Sci. USA 1993, 90: 11583-11587).
  • the human gene of the present invention has 98% homology with the mouse glutamine enrichment factor gene QRF-1 at the protein level, but contains 506 amino acids, and contains a characteristic domain of the HNF / fkh protein family, HNF / fkh domain (a DNA protein binding domain). Based on the above points, the new gene of the present invention is considered to be a full-length human glutamine enrichment factor gene and named hQRF-l. It is inferred that it is similar to QRF-1, is a member of the HNF / fkh protein family, and has the same biological function.
  • 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 glutamine enrichment factor 56.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human glutamine enrichment factor 56.
  • Another object of the present invention is to provide a method for producing human glutamine enrichment factor 56.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human glutamine enrichment factor 56.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention-human glutamine enrichment factor 56.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human glutamine enrichment factor 56.
  • a novel isolated human glutamine enrichment factor 56 is provided.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof , Or an active fragment thereof, or an active derivative or analog thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • a polynucleotide encoding the isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 98 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human glutamine enrichment factor 56; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 27-1547 in SEQ ID NO: 1; and (b) a sequence having 1-1580 in SEQ ID NO: 1 Sequence of bits.
  • Fig. 1 is a comparison diagram of amino acid sequence homology between the inventor's glutamine enrichment factor 56 and the mouse glutamine enrichment factor gene QRF-1.
  • the upper sequence is human glutamine enrichment factor 56 (hQRF-l), and the lower sequence is mouse glutamine enrichment factor gene QRF-1.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human glutamine enrichment factor 56. 45.6 kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated human glutamine enrichment factor 56 means that human glutamine enrichment factor 56 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 glutamine enrichment factor 56 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. Purity of human glutamine enrichment factor 56 polypeptide Can use amino acid sequence analysis.
  • the present invention provides a novel polypeptide-human glutamine enrichment factor 56, 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. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human glutamine enrichment factor 56.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human glutamine enrichment factor 56 of the present invention.
  • a fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by the genetic code; or ( ⁇ ) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ) like this One, in which 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 to the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence) As explained herein, 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 polynucleotide sequence of 1,580 bases in length, and its open reading frame (27-1547) encodes 506 amino acids.
  • the polynucleotide of the present invention is a full-length human glutamine enrichment factor 56 gene.
  • 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: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2, but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; The coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequences) of the mature polypeptide and non-coding sequences.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes 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.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) added during hybridization Use a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the identity between the two sequences is at least 95% or more, More preferably, hybridization does not occur until 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human glutamine enrichment factor 56.
  • 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 glutamine enrichment factor 56 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 DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating 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.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determining the level of the transcript of human glutamine enrichment factor 56; (4) Detecting the protein product of gene expression by immunological technology or measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and has a length of at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect protein products expressed by the human glutamine enrichment factor 56 gene expression protein.
  • ELISA enzyme-linked immunosorbent assay
  • 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 used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human glutamine enrichment factor 56 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding a human glutamine enrichment factor 56 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 adenovirus, reverse transcription, as is well known in the art. Virus or other vector.
  • 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, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human glutamine enrichment factor 56 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the 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. 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 the promoter to enhance gene transcription. 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. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli, etc.
  • 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 etc.
  • a polynucleotide encoding human glutamine enrichment factor 56 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf; animal cells such as CHO, COS or Bowes melanoma cells.
  • Transforming a host cell with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence This can be done 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 & 2 method, the steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human glutamine enrichment factor 56 (Science, 1984; 224: 1431). Generally, the following steps are taken:
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. 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.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, can be used to treat, for example, cancer, cardiovascular diseases, neurological diseases, immune diseases, inflammation and the like.
  • the polypeptide of the present invention can be directly used to promote cell proliferation, for example, contacting HQRF-1 with cardiomyocytes or neural cells to promote the repair of these cells. This is particularly useful for diseases such as myocardial infarction and ischemic stroke caused by myocardial ischemia.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human glutamine enrichment factor 56.
  • Agonists enhance biological functions such as human glutamine enrichment factor 56 to stimulate cell proliferation, and antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing human glutamine enrichment factor 56 can be cultured with labeled human glutamine enrichment factor 56 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human glutamine enrichment factor 56 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • Antagonists of human glutamine enrichment factor 56 can bind to human glutamine enrichment factor 56 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot function biological functions.
  • human glutamine enrichment factor 56 When screening compounds as antagonists, human glutamine enrichment factor 56 can be added to the bioanalytical assay, and the compound can be determined by measuring the effect of the compound on the interaction between human glutamine enrichment factor 56 and its receptor Whether it is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Peptide molecules capable of binding to human glutamine enrichment factor 56 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally, 56 molecules of human glutamine enrichment factor should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human glutamine enrichment factor 56 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 by injecting human glutamine enrichment factor 56 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's Adjuvant, etc.
  • Techniques for preparing monoclonal antibodies to human glutamine enrichment factor 56 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • 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 glutamine enrichment factor 56.
  • Antibodies against human glutamine enrichment factor 56 can be used in immunohistochemical techniques to detect human glutamine enrichment factor 56 in biopsy specimens.
  • Monoclonal antibodies that bind to human glutamine enrichment factor 56 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 glutamine enrichment factor 56 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol crosslinker such as SPDP, and toxin is bound to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human glutamine enrichment factor 56 positive Cell.
  • the antibodies in the present invention can be used to treat or prevent diseases related to human glutamine enrichment factor 56.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human glutamine enrichment factor 56.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human glutamine enrichment factor 56. These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human glutamine enrichment factor 56 detected in the test can be used to explain the importance of human glutamine enrichment factor 56 in various diseases and to diagnose the role of human glutamine enrichment factor 56. disease.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry.
  • human glutamine enrichment factor 56 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 glutamine enrichment factor 56.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human glutamine enrichment factor 56 to inhibit endogenous human glutamine enrichment factor 56 activity.
  • a mutated human glutamine enrichment factor 56 may be a shortened human glutamine enrichment factor 56 that lacks a signaling functional domain. Although it can bind to a downstream substrate, it lacks signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human glutamine enrichment factor 56.
  • 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 glutamine enrichment factor 56 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human glutamine enrichment factor 56 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human glutamine enrichment factor 56 can be packaged into liposomes and transferred into cells.
  • 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 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 glutamine enrichment factor 56 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes a specific RA. 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 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 RA 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 is used instead of the phosphodiester bond to link the ribonucleosides.
  • Polynucleotides encoding human glutamine enrichment factor 56 can be used in conjunction with human glutamine enrichment factor 56 Diagnosis of related diseases.
  • the polynucleotide encoding human glutamine enrichment factor 56 can be used to detect the expression of human glutamine enrichment factor 56 or abnormal expression of human glutamine enrichment factor 56 in a disease state.
  • a DNA sequence encoding human glutamine enrichment factor 56 can be used to hybridize biopsy specimens to determine the expression of human glutamine enrichment factor 56.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • 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.
  • Human glutamine enrichment factor 56 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect human glutamine enrichment factor 56 transcription products.
  • Detection of mutations in the human glutamine enrichment factor 56 gene can also be used to diagnose human glutamine enrichment factor 56-related diseases.
  • Human glutamine enrichment factor 56 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human glutamine enrichment factor 56 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. Therefore, Northern blotting and Western blotting can be used to indirectly determine the presence or absence of mutations in a gene.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for labeling chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35 bp) are prepared based on the cDNA, and the sequences can be located on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones 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 To correlate with genetic map data. These data can be found, for example, in V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, 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 20 kb correspond to one gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients 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 manufacture, use, or sell them.
  • the polypeptide of the present 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 glutamine enrichment factor 56 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human glutamine enrichment factor 56 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. Examples
  • 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 (Qiegen). 2ug poly (A) mRNA Transcribed to form cDNA.
  • the Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragments into the multicloning site of the pBSK (+) vector (Clontech) to transform DH5a bacteria (purchased from Promega) to form a cDNA library.
  • Dye terminate cycle reaction sequencing kit Perkin-Elmer
  • ABI 377 automated sequencing protocol Perkin-Elmer
  • the 0257b01 clone contains a full-length cDNA of 1580bp (as shown in Seq ID NO: 1), and has a 1521bp open reading frame (ORF) from 27bp to 1547bp, encoding a new protein containing 506bp (such as Seq ID NO: 2).
  • ORF open reading frame
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer.
  • PCR amplification was performed with the following primers:
  • Primer2 5 '-ATCTTCATTCTCGGGGTTGGCC-3' (SEQ IDNO.4)
  • Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions 50 mmol / L KCl, 10 mmol / L Tris-Cl, (pH 8.5), 1.5 mmol / L MgC12, 200 mol / L dNTP, lOpmol primer, 1U Taq DNA in a 50 ⁇ 1 reaction volume Polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C, 30sec; 72 ° C 2min.
  • ⁇ -actin was set as a positive control and template blank was set 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. DNA sequence analysis results indicate PCR The DNA sequence of the product is exactly the same as l-1580bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of HQRF-1 gene expression
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0) were used to hook the tissue into the ground, and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) Centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • Electrophoresis was performed on a 1.2% agarose gel containing 2 g of RNA on 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-ImM EDTA-2.2M formaldehyde. Then transferred to On a nitrocellulose membrane. A- 32 P dATP was used to prepare a 32 P-labeled DNA probe by a random primer method.
  • the DNA probe used was the PCR-amplified HQRF-1 coding region sequence (from ⁇ password ⁇ BP start position to the termination codon position ⁇ 1 ⁇ ⁇ ) the 32 P- labeled probes (about 2xl0 6 cpm / ml) and transferred to nitrocellulose membrane in RNA - hybridization solution overnight at 42.C
  • the solution contains 50% formamide-25mM KH 2 PO4 (pH7.4)-5xSSC-5xDenhardt's solution and 20 ( ⁇ g / ml salmon sperm DNA.
  • the filter is placed in lxSSC-0.1% SDS at 55 ° Wash for 30 min at C. Then, analyze and quantify with Phosphor Imager.
  • Primer 3 5 '-CAGGGATCCGATGCAGGTGGCTACCCAGCAGTTGG-3' (Seq ID No 5)
  • Primer4 5, -CCGAAGCTTTTCTCCATGTCCTCGTTTACTGGT -3, (Seq ID No 6)
  • the 5 'ends of these two primers contain BamHl and UindlU restriction sites, respectively
  • the coding sequences of the 5 and 3 'ends of the gene of interest are respectively followed by BamH1 and HindU restriction sites corresponding to the selective endonucleases on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Site.
  • PCR was performed using the PBS-0490B02 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-0490B02 plasmid in a total volume of 50 ⁇ 1, primers Primer-3 and Primer-4 were lOpmmol and Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles.
  • the amplified product and plasmid pET-28b (+) were double-digested with BamHI and Hindll, respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed with colibacillus Dh5ct (Promega) using the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), positive clones were selected by colony PCR and sequenced. . A positive clone ( ⁇ ET-0490B02) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (Novagen) using the calcium chloride method.
  • a peptide synthesizer (product of PE company) was used to synthesize the following HQRF-1 specific peptides:
  • This fragment is HQRF-1, a specific epitope.
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Immunochemistry, 1969; 6:43. Rabbits were immunized with 4 mg of the hemocyanin-polypeptide complex plus complete Freund's adjuvant, and 15 days later the hemocyanin-polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum.
  • Protein A-Sepharose was used to isolate total I gG from antibody-positive rabbit serum.
  • the peptide was bound to a cyanogen bromide-activated Sepharose 4B column, and anti-peptide antibodies were separated from total IgG by affinity chromatography.
  • the immunoprecipitation method demonstrated that the purified antibody could specifically bind to HQRF-1.
  • Example 7 HQRF-1 antisense oligonucleotide inhibits proliferation of HL-60 cells
  • AS-ODN Anti-sense oligonucleotides
  • S-ODN sense oligonucleotides
  • TETD Tetraethylthiuram disulfide
  • AS-PS-ODN 5 '-TAAGTAGGTCTTGAAGGTGCAGAGCAGGAG -3' (SEQ ID NO.8)
  • S-PS-ODN 5 '-CTCCTGCTCTGCACCTTCAAGACCTACTTA -3' (SEQ ID NO.9)
  • HL-60 cells (quoted from the Institute of Hematology, Chinese Academy of Sciences, Tianjin) with a concentration of 5 ⁇ 10 5 were divided into 3 groups and cultured in a 37 ° C, 5% CO 2 incubator: (1) The control group was pure HL- 60 cells; (2) HL-60 cells + AS-PS-ODN; (3) HL-60 cells + S-PS-ODN. The final concentration of AS-PS-ODN or S-PS-ODN is 20 ⁇ 8 / ⁇ 1. Cell death was measured by trypan blue staining after 24 hours of cell culture. The results showed that the cell death rate in the AS-PS-ODN group was 92.5%, while that in the control and S-PS-ODN groups was 5%. It is shown that the antisense oligonucleotide of HQRF-1 gene can effectively inhibit the proliferation of HL-60 cells.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un nouveau polypeptide, un facteur HOMO 56 riche en glutamine(Q), et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des cancers, des troubles cardio-vasculaires, des troubles du système nerveux, des troubles du système immunitaire et de diverses inflammations. L'invention concerne aussi les agonistes agissant contre le polypeptide et leur action thérapeutique ainsi que les applications de ce polynucléotide codant pour le nouveau facteur HOMO 56 riche en glutamine(Q).
PCT/CN2000/000310 1999-10-10 2000-10-08 Nouveau polypeptide, facteur homo 56 riche en glutamine(q), et polynucleotide codant pour ce polypeptide WO2001027148A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU77683/00A AU7768300A (en) 1999-10-10 2000-10-08 A novel polypeptide-homo glutamine(q)-rich factor 56 and polynucleotide encodingsaid polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN99116946.8 1999-10-10
CN 99116946 CN1292383A (zh) 1999-10-10 1999-10-10 一种新的多肽—人谷氨酰胺富集因子56和编码这种多肽的多核苷酸

Publications (1)

Publication Number Publication Date
WO2001027148A1 true WO2001027148A1 (fr) 2001-04-19

Family

ID=5279609

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2000/000310 WO2001027148A1 (fr) 1999-10-10 2000-10-08 Nouveau polypeptide, facteur homo 56 riche en glutamine(q), et polynucleotide codant pour ce polypeptide

Country Status (3)

Country Link
CN (1) CN1292383A (fr)
AU (1) AU7768300A (fr)
WO (1) WO2001027148A1 (fr)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 5 December 1997 (1997-12-05), Database accession no. A49395 *

Also Published As

Publication number Publication date
CN1292383A (zh) 2001-04-25
AU7768300A (en) 2001-04-23

Similar Documents

Publication Publication Date Title
WO2002051867A1 (fr) Nouveau facteur 8.8 polypeptidique liberant de la corticotrophine et polynucleotide codant ledit polypeptide
WO2001038522A1 (fr) Nouveau polypeptide, histone humaine h2a.21, et polynucleotide codant pour ce polypeptide
WO2001027148A1 (fr) Nouveau polypeptide, facteur homo 56 riche en glutamine(q), et polynucleotide codant pour ce polypeptide
WO2001031030A1 (fr) Nouveau polypeptide, phosphodiesterase 21 humaine de type acide sphingomyelinase, et polynucleotide codant pour ce polypeptide
WO2001029228A1 (fr) Nouveau polypeptide, caseine kinase humaine 48, et polynucleotide codant pour ce polypeptide
WO2001038375A1 (fr) Nouveau polypeptide, proteine a doigt de zinc 58 et polynucleotide codant pour ce polypeptide
WO2001030818A1 (fr) Nouveau polypeptide, proteine de liaison 33 a l'arn, et polynucleotide codant pour ce polypeptide
WO2001038379A1 (fr) Nouvelle proteine ribosomique humaine l23 a base d'un polypeptide et polynucleotide codant cette proteine
WO2001030837A1 (fr) Nouveau polypeptide, galectine 15, et polynucleotide codant pour ce polypeptide
WO2001038389A1 (fr) Nouvelle proteine ribosomique l14.22 a base d'un polypeptide et polynucleotide codant cette proteine
WO2001030840A1 (fr) Nouveau polypeptide, une proteine 57 a doigt de zinc, et polynucleotide codant pour ce polypeptide
WO2001031024A1 (fr) Nouveau polypeptide, threonine synthetase 71, et polynucleotide codant pour ce polypeptide
WO2001038370A1 (fr) Nouvelle sous-unite 49 de l'activateur de transcription polypeptidique et polynucleotide codant ce polypeptide
WO2001030827A1 (fr) Nouveau polypeptide, une proteine 56 a doigt de zinc, et polynucleotide codant pour ce polypeptide
WO2001027283A1 (fr) Nouveau polypeptide, proteine 16 de type transcriptase humaine inverse, et polynucleotide codant pour ce polypeptide
WO2001030839A1 (fr) Nouveau polypeptide, proteine humaine 33 de liaison a l'arn, et polynucleotide codant pour ce polypeptide
WO2001029229A1 (fr) Nouveau polypeptide, proteine humaine 20 de liaison de retinoblastome et polynucleotide le codant
WO2001029080A1 (fr) Nouveau polypeptide, une proteine humaine hmg-13, et polynucleotide codant pour ce polypeptide
WO2001030825A1 (fr) Nouveau polypeptide, facteur humain d'epissage 25, et polynucleotide codant pour ce polypeptide
WO2001031023A1 (fr) Nouveau polypeptide, proteine precurseur humaine 25 de la nucleoproteine ii, et polynucleotide codant pour ce polypeptide
WO2001030819A1 (fr) Nouveau polypeptide, tropomoduline humaine 39, et polynucleotide codant pour ce polypeptide
WO2001030826A1 (fr) Nouveau polypeptide, une serine/threonine kinase 29, et polynucleotide codant pour ce polypeptide
WO2001030834A1 (fr) Nouveau polypeptide, proteine 33 a doigt de zinc, et polynucleotide codant pour ce polypeptide
WO2001032863A1 (fr) Nouveau polypeptide, proteine humaine 12 associee a l'apoptose (sag), et polynucleotide codant pour ce polypeptide
WO2001030833A1 (fr) Nouveau polypeptide, proteine de regulation d'un nouveau cycle cellulaire 53, et polynucleotide codant pour ce polypeptide

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)