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WO2002032951A1 - Nouveau polypeptide, proteine a boite mads 44.33, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine a boite mads 44.33, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002032951A1
WO2002032951A1 PCT/CN2001/001443 CN0101443W WO0232951A1 WO 2002032951 A1 WO2002032951 A1 WO 2002032951A1 CN 0101443 W CN0101443 W CN 0101443W WO 0232951 A1 WO0232951 A1 WO 0232951A1
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Prior art keywords
polypeptide
polynucleotide
box protein
sequence
protein
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PCT/CN2001/001443
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU2002221433A priority Critical patent/AU2002221433A1/en
Publication of WO2002032951A1 publication Critical patent/WO2002032951A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, an F-box protein 44.33, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • the F-box protein family is a family of eukaryotic proteins with an increasing number of protein members.
  • the features of this family of proteins are that they all contain a motif consisting of about 40 amino acid residues, namely the F-box motif, the F-box
  • the protein family is also named for this [Bai C, Sen P, Hofman K, Ma L, et al.,
  • F-box proteins are key proteins that control the degradation of cell regulatory proteins [Pat ton E, Willems A, et al., Trends Genet 1998, 14: 6-14] [Koepp D , Ha Rper JW, et al, Cell 1999, 97:. 431-433] 0 in fact, F- box protein appears to pass a protein in a ligand [SCFs) four subunits. The other three subunits are Skpl, cullin and ROGI proteins.
  • SCFs ligand
  • Ubiquitin ligands guide ubiquitin binding enzymes to specifically bind to substrates, and proteins that specifically recruit substrates are different protein members in the F-box protein family. The high specificity of the substrate and the large number of F-box proteins required thereby prove that the F-box protein family is a large family of mammalian protein families.
  • F-box proteins there are 26 human F-box proteins in the F-box protein family. Some of these proteins contain WD-40 domains or leucine-rich repeats, while others contain different protein-protein interaction patterns or unrecognized motifs. Biologists refer to the F-box protein containing the WD-40 domain as the TO-40 domain Fbws protein.
  • the F-box protein containing leucine-rich repeats is the Fbls protein, and the rest contains zinc leucine fingers. Domain, ring finger domain, turn-loop-turn domain, and F-box protein with proline-rich motif and SH2 domain are Fbxs proteins [C. Cenciarelli, DS Chiaur, et al., Curr, Biol .1999, 9 (20), 1177-1179] o F-box protein family proteins have no similarity except for the presence of similarity in the upper domain.
  • the polypeptides of the present inventors and the Fbx22 protein in the F-box protein family have 93% identity and 93% similarity at the amino acid level, respectively, and have similar crust characteristics, both belong to the F-box protein family, It is named F-box protein 44.33, and it is speculated that it has similar biological functions.
  • the F-box protein 44.33 protein plays an important role in regulating important functions such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes. Therefore, in this field, it is necessary to identify more involved in these Process F-box protein 44.33 protein, especially this protein Amino acid sequence. Isolation of the novel F-box protein 44.33 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding F-box protein 44.33.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding an F-box protein 44.33.
  • Another object of the present invention is to provide a method for producing F-box protein 44.33.
  • Another object of the present invention is to provide an anti-F-box protein 44. 33 of the polypeptide of the present invention. Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the F-box protein 44. 33 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to the abnormality of F-box protein 44.33.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 12-1227 in SEQ ID NO: 1; and (b) a sequence having 1-2770 in SEQ ID NO: 1 Sequence of bits.
  • the present invention also relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the gene, including a transformed, transduced or transfected host cell; The method for preparing a polypeptide of the present invention by describing a host cell and recovering an expressed product is described.
  • the present invention also relates to an anti-oxidant capable of specifically binding to the polypeptide of the present invention.
  • the present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of F-box protein 44.33 protein, which comprises utilizing a polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of F-box protein 44.33 protein in vitro, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting The amount or biological activity of a polypeptide of the invention in a biological sample.
  • the present invention also relates to a pharmaceutical group of apricots, which comprises a polypeptide of the present invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of F-box protein 44.33.
  • Nucleic acid sequence means an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to the genome or Synthetic DNA or RNA, which can be single- or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the substituted amino acid has similar structural or chemical properties as the original amino acid, such as using leucine # "for isoleucine, and variants can also have non-conservative changes, such as Replace Glycine with Tryptophan.
  • “Deletion” means the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • “Insert” or “addition” means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more ammonia, amino acids, or nucleotides compared to the molecule that is present. Refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to natural, recombinant, or apricotinated proteins and fragments thereof in a suitable manner.
  • An "agonist” refers to a molecule that, when combined with F-box protein 44.33, causes the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind F-box protein 44.33.
  • Antagonist refers to a molecule that blocks or regulates the biological or immunological activity of F-box protein 44.33 when bound to F-box protein 44.33.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds F-box protein 44.33.
  • Regulation refers to a change in the function of F-box protein 44.33, including the increase or decrease in protein activity, changes in binding characteristics, and any other biological, functional, or immunological changes in F-box protein 44.33.
  • Substantially pure ' refers to other proteins, lipids, carbohydrates, or other substances with which it is not naturally associated. Those skilled in the art can purify F-box protein 44.33 using standard protein purification techniques. Substantially pure The F-box protein 44.33 can generate a single main band on a non-reducing polyacrylamide gel. The purity of the F-box protein 44.33 peptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides that are base-paired under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the reduced stringency allows non-specific binding, as the reduced stringency conditions require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Laser gene software package, DNASTAR, Inc, Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, I). G. and PM Shar (1988) Gene 73: 237 244). The Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: Number of residues matching between sequence A and sequence X 100 Number of residues in sequence A-number of interval residues in sequence A-number of interval residues in sequence B
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art, such as Jo t un He in (He in J., (1990) Method sin emzumo l ogy 183: 625-645) 0 "Similarity” refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • “Hang body” refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ) 2 and? , It can specifically bind to the F-box protein 44. 33 epitope.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen-binding region is replaced and changed to be more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is a solitary 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 F-box protein 44. 33 means that F-box protein 44. 33 is substantially free of other proteins, lipids, sugars, or other substances that appear to be associated with *. Those skilled in the art can use the standard 33. Quasi-protein purification technology purifies F-box protein 44.33. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of F-box protein 44. 33 polypeptide can be analyzed by amino acid sequence. The present invention provides a new polypeptide, F-box protein 44. 33, which is basically represented by SEQ II) NO: 2 1 Consisting of amino acid sequences.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptide of the present invention may be a naturally purified product, or a chemically synthesized product, or a prokaryotic or Produced in eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include or exclude initial methionine residues.
  • the invention also includes fragments, derivatives and analogs of F-box protein 44.33.
  • fragment As used in the present invention, the terms “fragment”, “derivative” and “analog” refer to a polypeptide that substantially maintains the same biological function or activity of the F-box protein 44.33 of the present invention.
  • the fragment, derivative or analog of the polypeptide of the present invention may be: (1) 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 (II) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III) such A type in which a mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide [Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a proteinogen 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 full-length polynucleotide sequence of 2770 bases and its open reading frame of 16-1227 encodes 403 amino acids. According to the amino acid sequence homology comparison, it was found that the polypeptide has 933 ⁇ 4 homology with the F-box protein, and it can be deduced that the box protein 44. 33 has a similar structure and function to the F-box protein.
  • the polynucleotides described herein can be in the form of DNA or RNA.
  • DNA forms include cDNA, genomics 1) NA or synthetic DNA.
  • DNA can be single-stranded or double-stranded. It can be a coding or non-coding chain.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SBQ 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 in the present invention, but differing from the coding region sequence shown in SEQ ID NO: 1.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; The coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (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.
  • a variant of this polynucleotide 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 present invention also relates to a polynucleotide that hybridizes to the sequence described above (the two sequences have at least 503 ⁇ 4, and preferably 70% identity).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.12SDS, 60 ° C; or ( 2 ) during hybridization Add denaturant, such as 503 ⁇ 4 (v / v) formamide, 0. calf serum / 0.1. /.
  • hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown by SBQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding F-box protein 44.33.
  • polypeptides and nucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the F-box protein 44. 33 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 the genome or c library to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect clones with common scab characteristics Polynucleotide fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) Isolating a double-stranded DNA sequence from genomic DNA: 2) Chemically synthesizing a DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical coupling of DNA sequences is often the method of choice. The more commonly chosen method is the separation of the c A sequences.
  • the standard method for isolating cl) NA is to isolate the mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or Phage cDNA library. There are many mature techniques for mRNA extraction. Kits are also commercially available (Qiagene). CDNA library is constructed in a conventional method (Sambrook, et al., Mo [ecu la r Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989) 0 commercially available cDNA library may also be obtained, such as Clontech Laboratories, Inc. Different ci) M libraries. 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 GDNA libraries by conventional methods. These methods include (fe is not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the level of the transcript of F-box protein 44.33; (4) Detection of gene-expressed protein products by immunological techniques or determination of 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 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.
  • DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product that detects the expression of the F-box protein 44.33 gene can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method of applying a PCR technique to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-GDM terminal rapid amplification method
  • the primers for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated 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 and the like. In order to obtain the full-length GDNA sequence, the sequencing must be repeated. Sometimes the cDNA sequences of multiple clones need to be determined in order to splice into full-length ci) NA sequences.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using the F-box protein 44.33 coding sequence, and a recombinant extraction method to produce the polypeptide of the present invention method.
  • the polynucleotide sequence encoding F-box protein 44.33 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide described in the Ming.
  • vector refers to a fine-plasmid, Bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors.
  • suitable carriers in the present invention include but are not limited to: T7 promoter-based expression vectors (Ros enberg, et al. Gene, 1987, 56: 125) expressed in bacteria; MSXND expressed in mammalian cells Expression vectors (Lee and Na thans, J Bio Ghem.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression loading is that it usually contains an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing an I) NA sequence encoding F-box protein 44.33 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sanibroook, et al. Mo l ecular Cl on ing, a Labora t ory Manua l, co ld Spr ing Harbor Labora tory. New York, 1989) .
  • the DNA sequence can be operably linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or tr p promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polytumor 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 recording for eukaryotic cell culture. Chromofluorescent 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 recording for eukaryotic cell culture. Chromofluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli, etc.
  • a polynucleotide encoding F-box protein 44.33 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a term for a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • "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 spp .; bacterial cells such as S.
  • 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 DNA can be harvested after the exponential growth phase and processed by the CaC method, and the procedures used are well known in the art.
  • the alternative is to use Mg (U 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, liposome packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant F-box protein 44. 33 (Scence, 1984; 224: 1431). Generally, the following steps are taken:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • Figure ⁇ is a comparison of the amino acid sequence homology of the F-box protein 44. 33 and F-box protein of the present invention.
  • the upper sequence is F-box protein 44. 33, and the lower sequence is F-box protein.
  • 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 the isolated F-box protein 44. 33. u
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • MRNA is formed by reverse transcription cDNA Quik mRNA Isolation Kit (Qiegene Co.) isolated from the total RNA poly (A) mRNA 0 2ug poly ( A) used.
  • a Smart cDNA cloning kit (purchased from C ontech) was used to insert the cDNA fragment into the multiple cloning site of the pBSK (+) vector (CLontedi) to transform DH5a.
  • the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • tiABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with a public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 1341 c 08 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 F-box protein 44.33 of the present invention and the protein sequence encoded by the F-box protein of the present invention were performed using the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], The same source search was performed in databases such as Genbank and Swissport.
  • the gene with the highest homology to the F-box protein 44.33 of the present invention is a known F-box protein, and the accession number encoded by the protein in Genbank is AF174602.
  • the results of protein homology are shown in Figure 1. The two are highly homologous and their identity is [ 31 ⁇ 2; similarity is 931.
  • Implementation ⁇ 3 The gene encoding F-box protein 44.33 is cloned by RT-PCR
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Priffler2 is the 3'-end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l of ol / L C1, 10 mmol / L Tris- in a reaction volume of 50 ⁇ i
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction.
  • the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) were added. ), Mix and centrifuge. 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.
  • RNA was synthesized by electrophoresis on a 1.23 ⁇ 4 agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2 M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation cc- 32 P dATP with 32 P- DNA probe labeled by the random primer method. The DNA probe used was the 44.33 coding region sequence (16bp to 1227bp) of the PCR-amplified F-box protein shown in FIG. 1.
  • a 32P-labeled probe (approximately 2 ⁇ 10 5 cpra / ml) and a nitrocellulose membrane to which RNA was transferred were placed in a solution at 42 ° C. C hybridization overnight, the solution contains 503 ⁇ 4 formamide_25mM H 2 P0 4 (pH7.4) -5 ⁇ SSC-5 ⁇ Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filters were placed in 1 x SSC-0.1% SI) S at 55. C: Wash 30 ⁇ . Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant F-box protein 44.33
  • Primer 3 5'- CCCCATATGATGGAGCCGGTAGGCTGC'TGCGGC- 3 '(Seq ID No: 5)
  • Prime: -4 5,-CCCGAATTCTTATTTAGATGACCCCAGATGTAT 3, (Seq ID No: 6)
  • the 5 ends of these two primers contain Ndel and EcoRI digestion sites, respectively, followed by the target gene 5, end And 3, coding sequences, Nde I and EGORI digestion sites correspond to selective endonuclease sites on the expression vector plasmid pET-28 b (+) (Nova gen product, Cat. No. 69865.3).
  • the PCR reaction was performed using pBS-1341G08 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS 1341c08 plasmid, primers Primer-3 and Primiei-4 were included in a total volume of 50 ⁇ 1, and 1 J was lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° G 2 min, a total of 25 cycles. Ndel and EcoRI were used to double digest the amplified product and plasmid pET 28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed with colibacillus DH5c by the calcium chloride method.
  • F-box protein 44.33 specific peptides were synthesized using a peptide synthesizer (product of PE company):
  • 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.
  • the probes can be used with genomic or ci) NA libraries of normal tissues or pathological tissues from different sources.
  • Cross In order to identify whether it contains the polynucleotide sequence of the present invention and detect a homologous polynucleotide sequence, the probe can also be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues. Or whether the expression in pathological tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method Acid sequence or a homologous polynucleotide sequence thereof.
  • Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same basic hybridization method after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the probes from the hybridization are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same or complementary to the polynucleotide SBQ ID NO: 1 of the present invention; the second type of probes are partially the same as this Polynucleotide of the invention SEQ ID NO: 1 Identical or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above requirements can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other unknown genomic sequences and their The complementary region is compared for homology. If the homology with the non-target molecular region is greater than 851 ⁇ 2 or there are more than 1 consecutive bases, then the primary probe should not be used;
  • Xiancheng selected and synthesized the following two probes after the analysis of the above aspects:
  • Probe 1 (probel), which belongs to the first type of probe, is completely homologous to the gene fragment of SEQ ID NO: 1 or 3 ⁇ 4 complement (4 lNt):
  • Probe 2 which belongs to the second type of probe, is equivalent to a good-for-mutation sequence (4lNt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane lutetium nitrate membrane
  • G X-ray autoradiography (press time depends on the radioactivity of the hybrid spot).
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HV infections and immune diseases. Wait"
  • the F-box protein family is a family of proteins in the body.
  • the proteins in this family all contain a motif consisting of about 40 amino acid residues, namely the F-box motif.
  • some F-box proteins are key proteins that control the degradation of cellular regulatory proteins. Its abnormal expression can cause disorder of protein metabolism in organisms, and then lead to related diseases.
  • the polypeptide of the present invention and the Fbx 22 protein in the F-box protein family are F-box proteins, which contain characteristic sequences of the protein family. Both have similar biological functions. Abnormal expression of the polypeptide in vivo can cause the protein Metabolic disorders, which in turn cause related diseases, including but not limited to:
  • disorders of protein metabolism can affect the following major physiological functions of proteins, and then cause related diseases. These diseases include, but are not limited to:
  • Muscle atrophy, weak limbs, body wasting, and severe cases can be manifested as "cachexia"; 2. Produce some physiologically active substances, such as hormones, anti-rest, amines, etc .:
  • Protein peptide hormone dysfunction can cause the following diseases:
  • Insulin and glucagon diabetes, hypoglycemia, etc .;
  • hypothalamus and pituitary hormones Giant disease, dwarfism, acromegaly, Cortisol syndrome (Cushing's syndrome), primary hyperaldosteronism, secondary chronic adrenal insufficiency, hyperthyroidism Hypothyroidism (stingle disease, juvenile hypothyroidism, adult hypothyroidism), male / female infertility, menstrual disorders (functional uterine bleeding, amenorrhea, polycystic ovarian syndrome, premenstrual stress syndrome) , Menopause syndrome), sexual development disorder, diabetes insipidus, improper antidiuretic hormone secretion syndrome, abnormal lactation, etc .;
  • parathyroid hormone hyperparathyroidism, hypoparathyroidism, etc .
  • Gastrointestinal hormones peptic ulcer, chronic digestion, chronic gastritis, etc .;
  • Arrhythmia shock, insanity, epilepsy, chorea, hepatic encephalopathy (norepinephrine, Y-aminobutyric acid, 5-hydroxytryptamine, glutamine), motion sickness, type 1 allergic disease (net Measles, hay fever, allergic rhinitis, skin allergies), peptic ulcer (histamine), hypercholesterolemia (taurine), tumors (polyamines), etc .;
  • Snoring purulent meningitis, pneumonia, bronchitis, otitis media, pus dermatosis, etc .;
  • Hemoglobinopathy anemia, jaundice, tissue hypoxia-induced organic acidemia
  • deficiency of various coagulation factors Deficiency (bleeding), muscle spasm, muscle forcing, muscle paralysis (actin), hyperlipoproteinemia Wait ⁇ ,
  • polypeptide of the invention and its antagonist, agonist and inhibitor can be directly used in the treatment of diseases such as diabetes, menstrual disorders, peptic ulcer, arrhythmia, blood, Epilepsy and other diseases.
  • This document also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) F-box protein 44.3.
  • Agonists increase F-box protein 44.33 to stimulate cell proliferation and other biological persuasion, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing F-box protein 44.33 can be cultured with labeled F-box protein 44.33 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of F-box protein 44.33 include antibodies, compounds, receptor deletions, and analogs that have been screened.
  • the antagonist of F-box protein 44.33 can bind to F-box protein 44.33 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 perform biological functions.
  • F-box protein 44.33 When screening compounds as antagonists, F-box protein 44.33 can be added to the bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between F-box protein 44.33 and its receptor. In the same way that compounds are used to screen compounds, resting deficiencies and analogs that function as antagonists can be screened. Polypeptide molecules capable of binding to F-box protein 44.33 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to the solid phase. When screening, generally 44.33 molecules of F-box protein should be 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 against the F-box protein 44.33 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • F-box protein 44.33 The production of polyclonal antibodies can be obtained by direct injection of F-box protein 44.33 into immunized animals (such as home immunity, mice, rats, etc.).
  • immunized animals such as home immunity, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's preparation.
  • F-box protein 44.33's monoclonal hang technology includes but is not limited to hybridoma technology (Kohier and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV-hybridization Tumor technology, etc.
  • the chimeric human antibody constant region and the variable region of non-human origin may be used in combination Pat some production techniques (Morrison et al, PNAS, 1985 , 81: 6851) 0 Ersi some production of single chain anti-break in the art (US Pat No. 4946778) can also be used to produce single chain antibodies against F-box protein 44.33.
  • the F-box protein 44.33 antibody can be used in immunohistochemistry to detect F-box protein 44.33 in biopsy specimens.
  • Monoclonal body bound to F-box protein 44.33 can also be labeled with radioisotopes and injected into the body
  • Hangxiu can also be used to design immunotoxins targeting a specific part of the body.
  • 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 F-box protein 44. 33
  • the antibodies can be used to treat or prevent diseases related to F-box protein 44.33.
  • An appropriate dose of antibody to the donor can stimulate or block the production or activity of F-box protein 44.33.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of F-box protein 44.33.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of F-box protein 44.33 detected in the test can be used to explain the importance of F-box protein 44.33 in various diseases and to diagnose diseases where F-box protein 44.33 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding F-box protein 44. 33 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 F-box protein 44.33. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated F-box protein 44.33 to inhibit endogenous F-box protein 44.33 activity. For example, a mutated F-box protein 44. 33 may be a shortened F-box protein 44. 33 lacking a signaling domain, although it can bind to downstream substrates and lack signaling activity.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of F-box protein 44.33.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated F-box protein 44.33 to inhibit endogenous F-box protein 44.33 activity.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of F-box protein 44.33.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding F-box protein 44.33 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding F-box protein 44. 33 can be found in the literature (Sambrook, et al.). 0 Another recombinant polynucleotide encoding F-box protein 44. 33 can be packaged. Transfer into liposomes 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 via a vector (such as a virus, phage, or plasmid) outside the cell, and then introducing the cell Transplant to Hugh and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit F-box protein 44.33 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RNA DNA and ribozymes can be obtained by any RNA or DNA synthesis technology. For example, solid-phase phosphate amide chemical synthesis technology 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 DM sequence is integrated downstream of the vector's RNA polymerase promoter.
  • it can be modified in a variety of ways, such as increasing the sequence length of two nucleotides, and the phosphorothioate or peptide bond is used instead of the phosphodiester bond for the ribonucleoside linkage.
  • Polynucleotide encoding F-box protein 44. 33 can be used to discontinue diseases related to F-box protein 44. 33. Polynucleotide encoding F-box protein 44. 33 can be used to detect F-box protein 44. 33 Expression or the abnormal expression of F-box protein 44. 33 in the disease state. For example, the DNA sequence encoding F-box protein 44. 33 can be used to hybridize biopsy specimens to determine the expression of F-box protein 44. 33. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly known 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 (Microarray) or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • F-box protein 44.33 specific primers can also be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect the transcription product of F-box protein 44.33.
  • Detection of mutations in the F-box protein 44 33 gene can also be used to diagnose F-box protein 44. 33-related diseases lendF-box protein 44. 33 mutations include forms that are similar to normal wild-type F-box protein 44. 33 DNA sequences Specific point mutations, translocations, deletions, recombinations, and any other abnormalities, etc. Mutations can be detected using well-known techniques such as Souter thern blotting, DNA sequence analysis, PCR, and in situ hybridization. In addition, mutations may affect protein expression Therefore, Nor thern blotting and Wes tern 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 of a human chromosome and can hybridize with it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few staining markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35 bp) are prepared based on GDNA, and the sequences can be mapped on chromosomes. Then, these primers are used for PGR screening of somatic hybrid cells containing individual human chromosomes. Hybrid cells based on the human gene of the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific staining cells.
  • oligonucleotide primers of the present invention a similar method can be used to achieve sublocalization by using a set of slices from a specific chromosome or a large number of genomic clones.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, staining with labeled flow sorting, prescreening and hybrid preselection to construct chromosome-specific cDNA library.
  • 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 observed in any normal period, the mutation may be the cause of the disease. Comparing diseased and non-diseased individuals usually involves first looking for changes in scabs in the chromosomes, such as defects or translocations that are visible at the chromosomal level or detectable with GDM sequence-based PGR. 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 Ability and every 2 Okb 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 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 incorporating one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • instructional instructions given by government regulatory agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government regulatory agencies that produce, use, or sell .
  • 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.
  • F-box protein 44. 33 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of F-box protein 44.33 to be administered to a patient will depend on many ⁇ , such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine à boîte MADS 44.33, et un polynucléotide codant 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 du diabète, des troubles de la menstruation, d'ulcères gastroduodénaux, d'arythmies, d'anémies et d'épilepsies. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine à boîte MADS 44.33.
PCT/CN2001/001443 2000-09-22 2001-09-21 Nouveau polypeptide, proteine a boite mads 44.33, et polynucleotide codant ce polypeptide WO2002032951A1 (fr)

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CN00125325.5 2000-09-22
CN 00125325 CN1345732A (zh) 2000-09-22 2000-09-22 一种新的多肽——f－盒蛋白44.33和编码这种多肽的多核苷酸

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999018989A1 (fr) * 1997-10-16 1999-04-22 Baylor College Of Medicine Proteines et genes a sequence f
WO2000012679A1 (fr) * 1998-08-28 2000-03-09 New York University Nouvelles ubiquitine ligases utiles comme cibles therapeutiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999018989A1 (fr) * 1997-10-16 1999-04-22 Baylor College Of Medicine Proteines et genes a sequence f
WO2000012679A1 (fr) * 1998-08-28 2000-03-09 New York University Nouvelles ubiquitine ligases utiles comme cibles therapeutiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CURR. BIOL., vol. 9, no. 20, 21 October 1999 (1999-10-21), pages 1177 - 1179 *

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