[go: up one dir, main page]

WO2001031030A1 - Nouveau polypeptide, phosphodiesterase 21 humaine de type acide sphingomyelinase, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, phosphodiesterase 21 humaine de type acide sphingomyelinase, et polynucleotide codant pour ce polypeptide Download PDF

Info

Publication number
WO2001031030A1
WO2001031030A1 PCT/CN2000/000386 CN0000386W WO0131030A1 WO 2001031030 A1 WO2001031030 A1 WO 2001031030A1 CN 0000386 W CN0000386 W CN 0000386W WO 0131030 A1 WO0131030 A1 WO 0131030A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
phosphodiesterase
similar
human acid
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2000/000386
Other languages
English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Bioroad Gene Development Ltd
Original Assignee
Shanghai Bioroad Gene Development 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 Bioroad Gene Development Ltd filed Critical Shanghai Bioroad Gene Development Ltd
Priority to AU13784/01A priority Critical patent/AU1378401A/en
Publication of WO2001031030A1 publication Critical patent/WO2001031030A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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 phosphodiesterase 21 similar to human acid sphingomyelinase, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
  • Apoptosis is a common phenomenon in living organisms, and usually occurs in hormone-dependent tissues, such as lymphocytes, thymocytes, liver cells, skin, and cells during embryogenesis. Apoptosis is a physiological control mechanism of cells. In this way, cells effectively control their own expression in various tissues. Apoptosis is accomplished by a series of regulatory factors. The abnormal expression of some of these regulatory factors will lead to the failure of programmed cell death, which will seriously lead to the excessive proliferation of some tumor cells, which will cause some related malignant diseases.
  • lymphoblastoid cells of patients with lipoidosis are different from normal lymphoblastoid cells. They cannot effectively activate the acid sphingomyelinase activity, so that the lymphoblastoid cells of this type of patients cannot aggregate GD3 ganglioside ( A downstream regulator of ceramide-mediated cell death). Therefore, the absence of acid sphingomyelinase in living organisms will lead to the failure of programmed cell death, which will cause various diseases related to it, such as the abnormal proliferation of some tumor cells, which will cause various malignant diseases related to it. And the overexpression of this enzyme will cause some tissue cells to die faster, which will lead to premature aging of some tissues, and will also cause some organs to fail.
  • the new human gene of the present invention has a high degree of homology with a phosphodiesterase similar to human acid sphingomyelinase, and has only 10 more N-terminus than a phosphodiesterase similar to human acid sphingomyelinase.
  • Signal peptide consisting of three amino acids. Therefore, it is a new phosphodiesterase similar to human acid sphingomyelinase, and it is named as phosphodiesterase 21 similar to human acid sphingomyelinase. This enzyme is similar to acid sphingomyelin diesterase and has similar functional characteristics.
  • 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 a phosphodiesterase 21 similar to human acid sphingomyelinase.
  • Another object of the present invention is to provide a method for producing a phosphodiesterase 21 similar to human acid sphingomyelinase.
  • Another object of the present invention is to provide an antibody against a phosphodiesterase 21 similar to the human acid sphingomyelinase polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the phosphodiesterase 21 similar to the human acid sphingomyelinase polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of phosphodiesterase 21 similar to human acid sphingomyelinase.
  • a novel isolated human acid sphingomyelinase-like phosphodiesterase 21 is provided.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2, or Its conservative variant polypeptide, or its active fragment, or its active derivative, analog.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 99 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding a phosphodiesterase 21 similar to the above-mentioned human acid sphingomyelinase; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • the sequence of the polynucleotide is one selected from the group consisting of: (a) a There is a sequence at positions 68-631 in SEQ ID NO: 1; and (b) a sequence at positions 1-939 in SEQ ID NO: 1.
  • a vector containing the above polynucleotide, and a host cell transformed or transduced by the vector or a host cell directly transformed or transduced by the above polynucleotide are provided.
  • Fig. 1 is a comparison diagram of the amino acid sequence homology of a phosphodiesterase 21 similar to the human acid sphingophosphatase and a phosphodiesterase similar to the human acid phosphatase of the present invention.
  • the upper sequence is a phosphodiesterase 21 similar to human acid sphingomyelinase
  • the lower sequence is a phosphodiesterase similar to human acid phosphatase.
  • 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 phosphodiesterase 21 similar to human acid sphingomyelinase. 21 kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human acid sphingomyelinase-like phosphodiesterase 21 means that human acid sphingomyelinase-like phosphodiesterase 21 is substantially free of other proteins, lipids, Sugars or other substances. Those skilled in the art can purify human acid sheath phosphatase-like phosphodiesterase 21 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human acid sphingomyelin-like phosphodiesterase 21 peptides can be analyzed by amino acid sequence analysis.
  • the present invention provides a new polypeptide, a phosphodiesterase 21 similar to human acid sphingomyelinase, 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 polypeptide of the present invention may be a naturally purified product, or a chemically synthesized product, or a recombinant technology from a prokaryotic or eukaryotic host (eg, bacteria, yeast, (Such as plants, insects, and mammalian cells).
  • a prokaryotic or eukaryotic host eg, bacteria, yeast, (Such as plants, insects, and mammalian cells.
  • the polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • Polypeptides of the invention may
  • the present invention also includes fragments, derivatives and analogs of phosphodiesterase 21 similar to human acid sphingomyelinase.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the phosphodiesterase 21 similar to the human acid sphingosphatases of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ )
  • Such a polypeptide sequence in which the mature polypeptide is fused with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence)
  • such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 939 bases in length and its open reading frame (68-631) encodes 187 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 99% homology with a phosphodiesterase similar to human acid phosphatase. It can be inferred that the phosphodiesterase 21 similar to human acid sphingomyelinase has human Acid phosphatase is similar in structure and function to phosphodiesterase.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDM, 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 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.
  • Polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) A denaturant was added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1 ° /. Fi co l l, 42.
  • Hybridization occurs only when the identity between the two sequences is at least 95% or more, and more preferably 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding phosphodiesterase 21, which is similar to human acid sphingomyelinase.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding a phosphodiesterase 21 similar to human acid sphingomyelinase of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) Isolation of double-stranded DNA from genome D Sequence; 2) chemically synthesize a DNA sequence to obtain 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 separation of cDM sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua 1, Cold Spruing 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.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DM or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of phosphodiesterase 21 similar to human acid sphingomyelinase The level of transcripts; (4) Detecting protein products expressed by genes by immunological techniques or by 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 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 the phosphodiesterase 21 gene similar to human acid sphingomyelinase can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). )Wait.
  • a method (Sa iki, et al. Sc; 1985; 230: 1350-1354) using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • Polynucleotide sequences of the gene of the present invention obtained as described above, or various DNA fragments can be used It is measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing 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 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 phosphodiesterase 21 coding sequence similar to human acid sphingomyelinase, and produced by recombinant technology A method of a polypeptide according to the invention.
  • a polynucleotide sequence encoding a phosphodiesterase 21 similar to human acid sphingomyelinase 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 (Rosenberg, et al.
  • any plasmid and vector can be used to construct recombinant expression vectors.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, 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. Examples to mention These 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 adenoviral 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 a phosphodiesterase 21 similar to human acid sphingomyelinase or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a polynucleotide containing the polynucleotide or the recombinant vector.
  • Genetically engineered host cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • 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 absorbing DM may be harvested after exponential growth phase, treated with CaC l 2 method used in steps well known in the art. The alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant phosphodiesterase 21 similar to human acid sphingomyelinase (Sc ience, 1984; 224: 1431). Generally speaking, there are the following steps:
  • the medium used in the culture may be selected from various Conventional medium. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Phosphodiesterases similar to acid sphingomyelinase have been cloned from mice and humans. This enzyme is similar to acid sphingomyelinase and catalyzes the hydrolysis of sphingomyelin to ceramide in vivo. The abnormal expression or inactivation of this enzyme will lead to the failure of programmed cell death, which will cause cell proliferation diseases, such as lipoidosis, pulmonary eosinophilia and so on. These cell proliferations are often accompanied by cell atypia, which can be further transformed into tumor cells, which can cause various related malignant diseases.
  • the enzyme and its fragments or derivatives can be used to treat various diseases such as cell proliferation and tumors caused by its abnormal expression.
  • diseases Including but not limited to the following, lipocytosis, malignant lymphoma (such as lymphatic reticulum, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), malignant histiocytosis, medulla Cell tumors, meningiomas, glioblastomas, acoustic neuromas, angiogenic tumors, pituitary adenomas, juxtaglomerular cell tumors, polycystic kidney tumors, seminoma, teratomas, testicular mesenchymal cells Tumor, endometrial stromal tumor, hydatidiform mole, ovarian tumor, breast fibro
  • malignant lymphoma such as lymphatic reticulum, malignant lymphoma, Hodgkin's
  • the human acid sphingomyelinase-like phosphodiesterase 21 of the present invention is expressed during embryogenesis.
  • Abnormalities can also be used to treat various developmental disorders caused by them. These diseases include but are not limited to the following: spina bifida, craniocerebral fissure, anencephaly, encephalocele, foramen deformity, Down syndrome, congenital Hydrocephalus, aqueduct malformation, cartilage hypoplasia, dwarfism, spinal epiphyseal dysplasia, pseudochondral dysplasia, Langer-G i ed i on syndrome, funnel chest, gonad hypoplasia, congenital adrenal hyperplasia, Upper urethral fissure, cryptorchidism, short stature syndrome such as Conrad i syndrome and Danbo l t C l os s syndrome, congenital glaucoma or cataract, congenital lens abnormality, congenital blepharoplasia,
  • human acid sphingomyelinase-like phosphodiesterase 21 of the present invention is overexpressed, it will also cause some cells to die excessively, that is, cause atrophy of some tissues or lose normal biological functions.
  • diseases include but are not limited to the following , Hypothyroidism, adrenal insufficiency, gonadal atrophy, functional atrophy of brain, heart, liver, skin, bone and other tissues.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human acid sheath phosphatase-like phosphodiesterase 21.
  • Agonists enhance human acid sphingomyelinase-like phosphodiesterase 21 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing a phosphodiesterase 21 similar to human acid sphingomyelinase can be cultured in the presence of a drug with a phosphodiesterase 21 similar to labeled human acid sphingomyelinase. . The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of phosphodiesterase 21 similar to human acid sphingomyelinase include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of phosphodiesterase 21 similar to human acid sphingomyelinase can bind to and eliminate functions of phosphodiesterase 21 similar to human acid sphingomyelinase, or inhibit the production of the polypeptide, or interact with the polypeptide The active site binding prevents the polypeptide from performing biological functions.
  • phosphodiesterase 21 similar to human acid sphingosphatase can be added to the bioanalytical assay.
  • 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 phosphodiesterase 21 similar to human acid sphingomyelinase can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase.
  • human acid sphingomyelin Esterase-like phosphodiesterase 21 molecules are labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against a phosphodiesterase 21 epitope similar to human acid sphingomyelinase. 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 directly injecting immunized animals (such as rabbits, mice, rats, etc.) with a phosphodiesterase 21 similar to human acid sphingomyelinase.
  • immunized animals such as rabbits, mice, rats, etc.
  • a phosphodiesterase 21 similar to human acid sphingomyelinase A variety of adjuvants can be used to enhance the immune response, including But it is not limited to Freund's adjuvant.
  • Techniques for the preparation of monoclonal antibodies similar to human acid sphingomyelase phosphodiesterase 21 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology , Human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that combine human constant regions with non-human-derived variable regions can be produced using existing techniques (Morrie 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 phosphodiesterase 21, which is similar to human acid sphingophosphatase.
  • Antibodies against phosphodiesterase 21, which is similar to human acid sphingomyelase, can be used in immunohistochemical techniques to detect phosphodiesterase 21, which is similar to human acid sphingomyelinase in biopsy specimens.
  • Monoclonal antibodies that bind to phosphodiesterase 21 similar to human acid sphingomyelinase 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 acid sphingomyelinase-like phosphodiesterase 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 acid sphingomyelase-like phosphates. Diesterase 21 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to phosphodiesterase 21 similar to human acid sphingomyelinase.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of phosphodiesterase 21, which is similar to human acid sphingomyelinase.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of phosphodiesterase 21 similar to human acid sphingomyelinase.
  • tests are well known in the art and include FISH assays and radioimmunoassay Determination.
  • Human acid sphingomyelinase-like phosphodiesterase 21 levels detected in the test can be used to explain the importance of human acid sphingomyelinase-like phosphodiesterase 21 in various diseases and to diagnose humans Diseases in which acid sphingomyelinase-like phosphodiesterase 21 functions.
  • 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.
  • Polynucleotides encoding phosphodiesterase 21 similar to human acid sphingomyelinase 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 phosphodiesterase 21, which is similar to human acid sphingosphate.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human acid sphingomyelinase-like phosphodiesterase 21 to inhibit endogenous human acid sphingomyelinase-like phosphodiesterase 21 active.
  • a variant human acid sphingomyelin-like phosphodiesterase 21 may be a shortened human acid sphingomyelin-like phosphodiesterase 21 lacking a signaling domain, although it may be similar to the downstream Substrate binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of phosphodiesterase 21, which is similar to human acid sphingomyelinase.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus, etc.
  • a polynucleotide encoding a phosphodiesterase 21 similar to human acid sphingophosphatase can be used to transfer a polynucleotide encoding a phosphodiesterase 21 similar to human acid sphingophosphatase to a cell Inside.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a phosphodiesterase 21 similar to human acid sphingomyelinase can be found in the existing literature (Sambrook, et al.).
  • recombinant polynucleotides encoding phosphodiesterase 21 similar to human acid sphingomyelinase 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 RM and DNA
  • ribozymes that inhibit phosphodiesterase 21 raRNA similar to human acid sphingomyelinase are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose a specific RNA, and its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense R and DNA and ribozymes can be obtained by any existing RNA or DNA synthesis technology. For example, the technology of solid phase phosphate amide synthesis of oligonucleotides has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of nucleic acid molecules, a variety of methods can be used to It is modified, such as increasing the sequence length on both sides, and the linkage between ribonucleosides uses phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • a polynucleotide encoding a phosphodiesterase 21 similar to human acid sphingomyelinase can be used for the diagnosis of diseases related to phosphodiesterase 21 similar to human acid sphingomyelinase.
  • Polynucleotides encoding phosphodiesterase 21 similar to human acid sphingomyelinase can be used to detect the expression of phosphodiesterase 21 similar to human acid sphingophosphatase or similar to human acid sphingomyelinase in disease states Abnormal Expression of Phosphodiesterase 21
  • a DNA sequence encoding a phosphodiesterase 21 similar to human acid sphing phosphatase can be used to hybridize biopsy specimens to determine the expression of phosphodiesterase 21 similar to human acid sphing phosphatase.
  • 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. Part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microaray) or a DNA chip (also called a "gene chip") for analyzing differential expression analysis of genes in tissues and gene diagnosis. .
  • Human acid sphingomyelinase-like phosphodiesterase 21-specific primers for R-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human acid sphingosidase-like phosphodiesterase 21 transcripts .
  • Detection of mutations in the phosphodiesterase 21 gene similar to human acid sphingomyelinase can also be used to diagnose diseases related to phosphodiesterase 21 similar to human acid sphingomyelinase.
  • Human acid sphingomyelinase-like phosphodiesterase 21 mutant forms include point mutations, translocations, deletions, recombination and recombination of phosphodiesterase 2 1 DNA sequences similar to normal wild-type human acid sphingomyelinase. Any other anomalies, etc. 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. Therefore, 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. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, the specific loci of each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome locations. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • the PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments. PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the 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 cD clones to metaphase chromosomes. allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization of cD clones to metaphase chromosomes.
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the 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 of the affected individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the 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 acid sphingomyelinase-like phosphodiesterase 21 is administered in an amount effective to treat and / or prevent a specific indication. The amount and dose range of human acid sphingomyelinase-like phosphodiesterase 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.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • the Quik mRNA Isolation Kit (Qiegene) was used to isolate poly (A) mT from total RNA. 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smar t cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5a. The bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0752h06 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the sequence of the phosphodiesterase 21 similar to the human acid sphingomyelinase of the present invention and the protein sequence encoded by the phosphodiesterase 21 were performed using the Blast program (Basic local alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], homology search was performed in Genbank, Swissport and other databases.
  • the gene with the highest homology of phosphodiesterase 21 similar to the human acid phosphatase of the present invention is a known human phosphodiesterase similar to phosphodiesterase, and the protein encoded by it is in Genbank The accession number is Y08136.
  • the protein homology results are shown in Figure 1.
  • Example 3 Cloning of a phosphodiesterase 21 encoding human acid sphingomyelinase by RT-PCR Genes were synthesized from fetal brain cell total RNA as a template, and oligo-dT was used as a primer for reverse transcription reaction to synthesize cD. After purification with Qiagene's kit, PCR was performed using the following primers:
  • Primer2 5'- AGATAAAGAATTCGCTTTATTG-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l reaction volume containing 50 ⁇ l / L KC1, 10 mmol / L Tris-Cl, (pH8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer , 1U Taq DNA 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.
  • the DNA sequence analysis results showed that the DM sequence of the PCR product was exactly the same as the 1-939bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human acid sphingomyelinase-like phosphodiesterase 21 gene expression:
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25m sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) are added. ) And 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.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM K 2 2 P 0 4 ( ⁇ 7 ⁇ 4) -5 xSSC-5 x Denhardt, s solution and 20 ( ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 minutes. Then, use Phosphor Imager was used for analysis and quantification.
  • Example 5 Recombinant human acid sphingomyelinase-like phosphodiesterase 21 in vitro expression, isolation and purification Based on the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, a For specific amplification primers, the sequence is as follows:
  • Primer 3 5'- CCCCATATGATGAGAGAATACTATAATGAGAAA-3 '(Seq ID No: 5)
  • Primer4 5'- CCCGGATCCCTAGTAATTGTGCTTTATATAAAG-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Ndel and BamHI restriction sites correspond to the selectivity on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • the PCR reaction was performed using pBS-0752h06 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-0752h06 plasmid contained in a total volume of 50 ⁇ l, primers Primer-3 and Primer-4 were lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into E. coli DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were screened by colony PCR method and sequenced. A positive clone (pET-0752h06) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • NH 2 -Met-Arg-Glu-Tyr-Tyr-Asn-G 1 u-Lys-Leu-I le-Asp-I l e-Phe-Gln-Lys-COOH SEQ ID NO: 7
  • 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, I bandnochemi s try, 1969; 6: 4. Rabbits were immunized with 1 ⁇ 2 g 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 antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to phosphodiesterase 21, which is similar to human acid sphingomyelinase.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un nouveau polypeptide, une phosphodiestérase 21 humaine de type acide sphingomyélinase, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la phosphodiestérase 21 humaine de type acide sphingomyélinase.
PCT/CN2000/000386 1999-10-28 2000-10-27 Nouveau polypeptide, phosphodiesterase 21 humaine de type acide sphingomyelinase, et polynucleotide codant pour ce polypeptide Ceased WO2001031030A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU13784/01A AU1378401A (en) 1999-10-28 2000-10-27 A novel polypeptide, a human acid sphingomyelinase-like phosphodiesterase 21 andthe polynucleotide encoding the polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN99119899.9 1999-10-28
CN99119899A CN1302897A (zh) 1999-10-28 1999-10-28 一种新的多肽——人酸性鞘磷酯酶相似的磷酸二酯酶21和编码这种多肽的多核苷酸

Publications (1)

Publication Number Publication Date
WO2001031030A1 true WO2001031030A1 (fr) 2001-05-03

Family

ID=5281190

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2000/000386 Ceased WO2001031030A1 (fr) 1999-10-28 2000-10-27 Nouveau polypeptide, phosphodiesterase 21 humaine de type acide sphingomyelinase, et polynucleotide codant pour ce polypeptide

Country Status (3)

Country Link
CN (1) CN1302897A (fr)
AU (1) AU1378401A (fr)
WO (1) WO2001031030A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001074900A3 (fr) * 2000-03-31 2002-05-02 Aventis Pharm Prod Inc Facteur inducteur du facteur nucleaire kb
US7041497B2 (en) 2000-03-31 2006-05-09 Aventis Pharmaceuticals Products Inc. Nuclear factor κB inducing factor
US11898175B2 (en) 2017-08-24 2024-02-13 Genzyme Corporation Treatment of abnormal bone conditions in acid sphingomyelinase deficiency patients

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 19 September 1996 (1996-09-19), "HSASML3A", Database accession no. Y08136.1 *
DATABASE EMBL [online] 19 September 1996 (1996-09-19), "MMASML3A", Database accession no. Y08135.1 *
GENOME RES., vol. 8, no. 11, November 1998 (1998-11-01), pages 1097 - 1108 *
NATURE, vol. 368, no. 646, 3 March 1994 (1994-03-03), 38, pages - 32 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001074900A3 (fr) * 2000-03-31 2002-05-02 Aventis Pharm Prod Inc Facteur inducteur du facteur nucleaire kb
US7041497B2 (en) 2000-03-31 2006-05-09 Aventis Pharmaceuticals Products Inc. Nuclear factor κB inducing factor
US7972844B2 (en) 2000-03-31 2011-07-05 Aventis Pharmaceuticals Inc. Nuclear factor κB inducing factor
US8623831B2 (en) 2000-03-31 2014-01-07 Aventis Pharmaceuticals Inc. Nuclear factor κB inducing factor
US8642739B2 (en) 2000-03-31 2014-02-04 Aventis Pharmaceuticals Inc. Nuclear factor κB inducing factor
US11898175B2 (en) 2017-08-24 2024-02-13 Genzyme Corporation Treatment of abnormal bone conditions in acid sphingomyelinase deficiency patients

Also Published As

Publication number Publication date
AU1378401A (en) 2001-05-08
CN1302897A (zh) 2001-07-11

Similar Documents

Publication Publication Date Title
WO2002006477A1 (fr) Nouveau polypeptide, topologie isomerase humaine 12.1, et polynucleotide codant ce polypeptide
WO2002026973A1 (fr) Nouveau polypeptide, une ribonucleoproteine nucleaire heterogene humaine 32.01, et un polynucleotide codant 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
WO2002026791A1 (fr) Nouveau polypeptide, serine kinase 212.98 specifique d'une proteine sr, et polynucleotide codant ce polypeptide
WO2001030837A1 (fr) Nouveau polypeptide, galectine 15, et polynucleotide codant pour ce polypeptide
WO2001031001A1 (fr) Nouveau polypeptide, facteur auxiliaire 28 du facteur de demarrage de la traduction, et polynucleotide codant pour ce polypeptide
WO2001030840A1 (fr) Nouveau polypeptide, une proteine 57 a doigt de zinc, et polynucleotide codant pour ce polypeptide
WO2001030818A1 (fr) Nouveau polypeptide, proteine de liaison 33 a l'arn, et polynucleotide codant pour ce polypeptide
WO2001030826A1 (fr) Nouveau polypeptide, une serine/threonine kinase 29, et polynucleotide codant pour ce polypeptide
WO2001038376A1 (fr) Nouveau polypeptide, proteine humaine a doigt de zinc 46, et polynucleotide codant pour ce polypeptide
WO2001038389A1 (fr) Nouvelle proteine ribosomique l14.22 a base d'un polypeptide et polynucleotide codant cette proteine
WO2001031024A1 (fr) Nouveau polypeptide, threonine synthetase 71, et polynucleotide codant pour ce polypeptide
WO2001031022A1 (fr) Nouveau polypeptide, arginyl arnt synthetase 44, 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
WO2001030838A1 (fr) Nouveau polypeptide, proteine g 17, et polynucleotide codant pour ce polypeptide
WO2001032698A1 (fr) Nouveau polypeptide, proteine humaine 16 associee aux maladies auto-immunes, et polynucleotide codant pour ce polypeptide
WO2001029075A1 (fr) Nouveau polypeptide, proteine 129 activant la proteine g, et polynucleotide codant pour ce polypeptide
WO2001030832A1 (fr) Nouveau polypeptide, proteine a doigt de zinc hkznf-23, et un polynucleotide codant pour ce polypeptide
WO2001030819A1 (fr) Nouveau polypeptide, tropomoduline humaine 39, et polynucleotide codant pour ce polypeptide
WO2001027283A1 (fr) Nouveau polypeptide, proteine 16 de type transcriptase humaine inverse, et polynucleotide codant pour ce polypeptide
WO2001030841A1 (fr) Nouveau polypeptide, proteine 60 a doigt de zinc, et polynucleotide codant pour ce polypeptide
WO2001030827A1 (fr) Nouveau polypeptide, une proteine 56 a doigt de zinc, et polynucleotide codant pour ce polypeptide
WO2001027285A1 (fr) Nouveau polypeptide, methyltransferase humaine dpb 41 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 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)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP