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WO2002020777A1 - Nouveau polypeptide, proteine myd24.09 de transmission de signaux cellulaires, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine myd24.09 de transmission de signaux cellulaires, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002020777A1
WO2002020777A1 PCT/CN2001/001117 CN0101117W WO0220777A1 WO 2002020777 A1 WO2002020777 A1 WO 2002020777A1 CN 0101117 W CN0101117 W CN 0101117W WO 0220777 A1 WO0220777 A1 WO 0220777A1
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Prior art keywords
polypeptide
polynucleotide
cell signaling
signaling protein
sequence
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU2002223367A priority Critical patent/AU2002223367A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a cell signaling protein MyD24.09, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing such polynucleotides and polypeptides. Background technique
  • MyD88 was first isolated and identified as a corresponding gene for spinal cord differentiation in mice (mye loid different iat ion pr imary response gene). It is activated in Ml spinal cord leukocytes induced by interleukin 6-terminated growth termination and terminal differentiation. [Oncogene 1996 Dec 5; 13 (11): 2467- 75]
  • MyD88 contains an N-terminal death doma in (DD) and a C-terminal Toll domain.
  • the DD domain is related to a 90-amino acid domain, which is found in proteins such as FAS / Apol / CD95 and TNF receptors, and is related to its cytotoxin-induced signaling. DD domains can combine with each other to form homo- or heterodimers, which are involved in protein-protein interactions.
  • the C-terminal To l l domain of MyD88 consists of 130 amino acid residues. This domain is homologous to the cytoplasmic signaling part of the transmembrane proteins Tol l and IL-1RI in Drosophila. It is also found in many different protein families. Most of these protein families are cell surface receptors. The Tol l structure itself does not contain the ability for complex signaling, but it can aggregate related proteins together to conduct signals.
  • MyD88 is a protein containing a cell signaling domain and should play a role in cell signaling. [: T Biol Chem 1998 May 15; 273 (20): 12203-9]
  • 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 cell signaling protein MyD24.09.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a cell signaling protein MyD24.09.
  • Another object of the present invention is to provide a method for producing a cell signaling protein MyD24.09.
  • Another object of the present invention is to provide a polypeptide-to-cell signaling protein directed to the polypeptide of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide-cell signaling protein MyD24.09 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 the cell signaling protein MyD24.09.
  • 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 295-954 in SEQ ID NO: 1; and (b) a sequence having 1-1384 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a screened protein that mimics, activates, antagonizes or inhibits cell signaling A method of MyD24.
  • 09 protein active compounds 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 disease susceptibility related to abnormal expression of a cell signaling protein MyD24.09 protein in vitro, comprising detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting The amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the manufacture of a medicament for treating ⁇ cancer, developmental disease or immune disease ⁇ or other diseases caused by abnormal expression of the cell signaling protein MyD24.09.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DM or RNA, they can be single-stranded 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 amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • Antagonist means that when combined with the cell signaling protein MyD24.09, an Molecules whose white matter changes to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate or any other molecule that can bind to the cell signaling protein MyD24.09.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of the cell signaling protein MyD24.09 when combined with the cell signaling protein MyD24.09.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to the cell signaling protein MyD24.09.
  • Regular refers to a change in the function of the cell signaling protein MyD24.09, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of the cell signaling protein MyD24.09. change.
  • substantially pure is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify the cell signaling protein MyD24.09 using standard protein purification techniques.
  • the substantially pure cell signaling protein MyD24.09 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the cell signaling protein MyD24. 09 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules 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 Nor thern 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 conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are the same or similar in a comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (La ser gene sof tware package, DNASTAR, Inc., Mad Son Wis.). The MEGALIGN program can compare two or more sequences (Higgins, D. G. and
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in emzumo logy 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RM 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.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and?, Which specifically bind to the epitope of the cell signaling protein MyD24.09. ,
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it 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 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 cell signaling protein MyD24. 09 means the cell signaling protein MyD24. 09 is substantially free of other proteins, lipids, carbohydrates, or other substances naturally associated with it. Those skilled in the art can purify the cell signaling protein MyD24.09 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the cell signaling protein MyD24. 09 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a cell signaling protein MyD24.09, 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, Synthetic polypeptides, preferably recombinant polypeptides.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of the cell signaling protein MyD24.09.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the cell signaling protein MyD24.09 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 full-length polynucleotide sequence of 1384 bases, and its open reading frames 295-954 encode 219 amino acids. According to the comparison of gene chip expression profiles, it was found that this peptide has a similar expression profile to the cell signaling protein MyD88, and it can be inferred that the cell signaling protein MyD24. 09 has a cell signaling protein
  • MyD88 has similar functions.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DM can be a coding chain or a non-coding chain.
  • the coding region sequence encoding the 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 having a sequence 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 of the mature polypeptide (and optionally the additional Plus coding sequences) 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.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.11 ⁇ 2SDS, 60 ° C; or ( 2 ) during hybridization Add denaturant, such as 50 ° /.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding the cell signaling protein MyD24.09.
  • 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 the cell signaling protein MyD24.09 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 DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the DM of the genome; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice. The more commonly used method is the separation of the CDM sequences.
  • the standard method for isolating cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage CDM library. There are many mature technologies for ffl RNA extraction. Kits are also available from commercial Way to get (Qiagene). CDM libraries are also commonly used (Sambrook, et al., Molecular Cloning, A Laboratory Manua, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • 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-DNA or DM-RM hybridization; (2) the appearance or loss of marker gene function; (3) determination of the level of the transcript of the cell signaling protein MyD24.09; ( 4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and 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 DM 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, fluorescein, or enzymes (such as alkaline phosphatase).
  • the protein product of the cell signaling protein MyD24. 09 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method (Sa iki, et al. Science 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 for PCR may 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 DM 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 cDM sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDM sequence of multiple clones in order to splice into a full-length cMA 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 the cell signaling protein MyD24.09 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding a cell signaling protein MyD24.09 can be inserted into a vector In order 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 a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • the MA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells.
  • Enhancers are cis-acting factors expressed by DM, 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, tumorigenic 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 cell signaling protein MyD24.09 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • insect cells such as flies S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl, the steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant cell signaling protein MyD24. 09 (Sc ience, 1984; 224: 1431). Generally there are the following steps:
  • 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
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the cell signaling protein MyD24.09 and the cell signaling protein MyD88 of the present invention.
  • the upper graph is a graph of the cell signal transduction protein MyD24. 09, and the lower graph is the graph of the cell signal transduction protein MyD88.
  • FIG 2 shows the polyacrylamide gel electrophoresis of the isolated cell signaling protein MyD24.09 (SDS-1)
  • Human fetal brain total MA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Using Quik mRNA Isolation Kit
  • a Smart cDNA cloning kit purchased from Clontech was used to insert the 00 fragment into a multi-cloning site of a pBSK (+) vector (Clontech) to transform DH5 ⁇ to form a cDNA library.
  • Dye terminate cycle reaction sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0051d06 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the results show that the full-length cDNA contained in the 0051d06 clone is 1384b P (as shown in Seq lDNO: 1), and has a 659bp open reading frame (0RF) from 295bp to 954b P , which encodes a new protein (such as Seq ID NO: 2 ).
  • This clone pBS-0051d06 and the encoded protein was named the cell signaling protein MyD24.09.
  • Example 2 Cloning of the gene encoding the cell signaling protein MyD24.09 by RT-PCR
  • CDM was synthesized using fetal brain cell total MA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5'-GGGGAGTGGCGTGCTGGGCGTGCG-3 '(SEQ ID NO: 3)
  • Primer2 5'-CAGTAGAAACTTATTTTTATTATC-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp; Pr imer2 is the 3′-end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions reaction volume containing 50 ⁇ 1 of 50mnio l / L KC1, 10mmo l / L Tr i s- Cl, (pH8 5.), 1. 5mmo l / L MgCl 2, 200 ⁇ mol / L dNTP, Opmo primer, 1U Taq DNA polymerase (Clontech). 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 0 ⁇ -act in was set as positive at the time of RT-PCR Controls and template blanks are negative controls.
  • the amplified product was purified using a QIAGEN kit and ligated to a PCR vector using a TA cloning kit (Invitrogen).
  • the sequence analysis result of A1 shows that the DNA sequence of the PCR product is exactly the same as that of 1 to 1384bp shown in SEQ ID NO: 1.
  • Example 3 Analysis of the expression of the cell signaling protein MyD24.09 gene by Northern blotting:
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RM was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7. 4)-5 x SSC- 5 x Denhardt, s solution and 200 ⁇ ⁇ / ⁇ 1 salmon sperm DNA. After hybridization, the filter was placed at 1 X SSC-0.1 ° /. Wash in SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant cell signaling protein MyD24.09
  • Pr imer 3 5'-CCCCATATGATGTATTTCAAACGTGCCGCTCGG -3 '(Seq ID No: 5)
  • Pr imer 5'-CCCGAATTCTCAAGGTGAGGTTGTGTCTCTGGC -3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI digestion respectively Site, followed by the coding sequence of the 5 'end and 3' end of the gene of interest, respectively, and the Ndel and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • the PCR reaction was performed using the pBS-00 5 ld06 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are: pBS- 0051d06 in a total volume of 50 ⁇ 1
  • the plasmid 10 pg, primers Primer-3 and Pi-imer-4 were divided into two parts; l is lpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1.
  • 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 the colibacillus DH5ct by the calcium chloride method, and cultured overnight in LB plates containing kanamycin (final concentration 3 () ⁇ ⁇ / ⁇ 1), and then positive clones were selected by colony PCR method and sequenced. Selected positive clones with the correct sequence (pET- 0051d06) the recombinant plasmid by the calcium chloride method to transform E. coli BL21 (DE3) P lySs (Novagen Co.).
  • the host strain BL21 (pET-0051d06) was 37 in LB liquid medium containing kanamycin (final concentration 30 ⁇ g / ml). C. Cultivate to logarithmic growth phase, add IPTG to a final concentration of 1 ol / L, and continue to cultivate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidine (6His-Tag). purified protein intracellular signaling protein MyD24.09 o by SDS- PAGE electrophoresis and a single band (FIG.
  • a peptide synthesizer (product of PE company) was used to synthesize the following cell signaling protein MyD24.09-specific peptides:
  • NH2-Met-Tyr-Phe-Lys-Arg-Ala-Ala-Arg-Ala-Phe-Pro-Val-Leu-Leu-Thr-C00H 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. Immunochemistry, 1969; 6: 43.
  • the rabbits were immunized with 4 mg of the above-mentioned blood: protein peptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin peptide 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.
  • Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit serum.
  • 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 the cell signaling protein MyD24.09.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in 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, Northern blotting, and copying methods. They all use the same steps to immobilize 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 unhybridized probes 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 as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same 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 from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes 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 conditions 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 known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe ⁇ , is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • Probes 1 3 ⁇ l Probe (0. 10D / 10 ⁇ 1), add 2 ⁇ IKinase buffer, 8-10 uCi ⁇ - 32 P- dATP + 2U Kinase, to make up to a final volume of 20 ⁇ 1.
  • Gene chip or gene microarray is a new technology currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass. And silicon, and then use fluorescence detection and computer software to compare and analyze the data. Analysis in order to achieve the purpose of fast, efficient and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases, such as genetic diseases .
  • the specific method steps have been reported in the literature, such as DeRis i, JL, Lyer, V. & Brown, P. 0 (1997) Science 278, 680-686. And Hel le, RA, Schema. , M., Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDMs were used as target DNA, including the polynucleotides of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500ng / ul after purification.
  • the spots were spotted on a sloped glass using a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ ⁇ .
  • the spotted slides were hydrated, dried, and cross-linked in a purple diplomatic instrument. After elution, the DM was fixed on the glass slide to prepare chips.
  • the specific method steps are widely reported in the literature.
  • the post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen), and separated by reverse transcription! ] Cy3dUTP (5-Amino-propargy 1-2'-deoxyur idine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP ( 5- Amino- propargy 2 '-deoxyur idine 5'-tr iphate cou led to Cy5 f luorescent dye, purchased from Amersham Phamacia Biotech Company, labeled with specific tissue (or stimulated cell line) mRNA in the body, and purified and prepared Probe.
  • Oligotex mRNA Midi Kit purchased from QiaGen
  • Cy3dUTP 5-Amino-prop
  • the probes from the above two tissues and the chips were respectively hybridized in a UniHyb TM Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature. Scanning was then performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 1024NT, scar formation fc growth factor stimulation, 1013HT, scar into fc without growth factor stimulation, 1G13HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, Cardiac cancer. Draw a graph based on these 18 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the cell signaling protein MyD24.09 and the cell signaling protein MyD88 according to the present invention have very similar expression profiles.
  • Industrial applicability is fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC,
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • the human MyD88 protein is a protein containing a cell signaling domain. In the body, it can aggregate related proteins to conduct signals. Its abnormal expression can cause the dysfunction of the cell signaling system (such as cAMP signaling pathway), and then lead to the occurrence of related diseases.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human MyD88 protein, and both have similar biological functions.
  • the polypeptide of the present invention is a protein containing a cell signaling domain in the body, and it can aggregate related proteins to conduct signals. Its abnormal expression can cause dysfunction of the cell signaling system (such as cAMP signaling pathway), and then lead to disease.
  • cAMP is widely found in prokaryotic and eukaryotic cells. It is an important regulatory factor in cells and has a wide range of regulatory activities at the protein level and gene DNA level. I. Regulation of metabolic processes.
  • the cAMP system activates and inhibits the activities of many key metabolic enzymes through protein kinases (PKA), controls the decomposition and synthesis of sugars through the amplification of cascade reactions, and activates lipases to modify and promote or inhibit lipolysis.
  • PKA protein kinases
  • disorders in this regulatory process can lead to the following diseases, including but not limited to In: Systemic multisystem vascular sclerosis (cardio-cerebral blood vessels, renal blood vessels: limb peripheral blood vessels), ophthalmic diseases (metabolic cataract, iris ciliary body inflammation), neurological diseases (hyperosmotic coma, hypoglycemic encephalopathy) Various infections (skin infections, reproductive system infections), fatty deposition diseases (fatty liver, fatty deposition cardiomyopathy, fatty deposition nephropathy) and related tumors (lipoma, lipoblastoma, liposarcoma), etc. 2. Regulation of hormones and nervous system.
  • adenylate cyclase ACa s e
  • cAMP adenylate cyclase
  • adrenocorticotropic hormones lead to increased synthesis of tooth hormones.
  • the synaptic membrane of nerve cells in the brain contains highly active PKA, which plays an important role in the synthesis, release and transmission of neurotransmitters. Disorders in this regulatory process can lead to the following diseases, including but not limited to:
  • Glucocorticoids cortisol: high / low blood sugar, muscle wasting, osteoporosis, delayed wound healing, infection, centripetal obesity, water poisoning (headache, convulsions, coma), mental disorders, etc .;
  • Mineralocorticoids aldosterone: edema, hypertension, high / low blood sodium (headache, convulsions, coma), high / low blood potassium (muscle paralysis, arrhythmia, renal failure, paralytic intestinal obstruction, drowsiness, Coma) etc;
  • Estrogen sexual developmental abnormalities, male feminization, menstrual disorders, lactation abnormalities, edema, hypertension, heart failure (water and sodium storage), menopausal syndrome, ovarian insufficiency, functional uterine bleeding, osteoporosis Tumors (endometrial cancer, breast cancer), etc .;
  • Progestogens dysmenorrhea, amenorrhea, endometriosis, threatened / habitual abortion, endometrial cancer, etc .;
  • Androgens sexual developmental abnormalities, virilization of women, anemia (stimulating bone marrow hematopoiesis), tumors (prostate cancer, breast cancer), edema, hypertension, etc .;
  • Frontal lobe dementia, personality changes (frontal frontal), strabismus, inability to write (back middle frontal gyrus), motor aphasia (back frontal lower back), olfactory loss (bottom of frontal lobe), paralysis of the limbs, Convulsions (central gyrus), etc .;
  • Parietal lobe sensory disturbance (central posterior gyrus), dyslexia (left corner gyrus), body image disorder (right parietal lobe) Wait;
  • Temporal lobe Hookback attack (anterior temporal lobe), sensory / amnestic aphasia (left temporal lobe), hearing impairment (rear superior temporal gyrus), etc .;
  • Occipital lobe hemianopia, hallucination, visual disagreement, etc .
  • Limb system emotional symptoms, memory loss, disturbance of consciousness, hallucinations, etc .; 3. Phosphorylation of membrane proteins.
  • PKA phosphorylates tubulin and other cells, and affects the movement of organelles within the cell and the cell growth cycle. This dysfunction can lead to the following diseases, including but not limited to: Common embryonic malformations
  • Cleft lip most common, with alveolar cleft and cleft palate), cleft palate, oblique facial cleft, cervical pouch, cervical fistula, etc .;
  • Absent limbs Transverse absent '(congenital short limbs): no arms, no forearms, no hands, no fingers, no legs, no toes
  • Limb differentiation disorder Absence of a certain muscle or muscle group, joint dysplasia, bone deformity, bone fusion, multi-finger (toe) deformity, and finger (toe) deformity, horse tellurium varus, etc .;
  • Thyroglossal duct cysts atresia or stenosis of the digestive tract, ileal diverticulum, umbilical fistula, congenital umbilical hernia, congenital agangliomegalo colon, impotence of anus, abnormal bowel transition, bile duct atresia, circular pancreas, etc
  • neural tube defects no cerebral malformations, spina bifida, spinal meningocele, hydrocephalous meningoencephalocele), hydrocephalus inside / outside the brain, etc .
  • Papilloma squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (carcinoma) [breast, thyroid], mucinous / serous cystadenoma (carcinoma) [ovarian], basal cell carcinoma [head and face Skin], (malignant) polytype adenoma [extending gland], papilloma, transitional epithelial cancer [bladder, renal pelvis], etc .;
  • Malignant lymphoma [Neck, mediastinum, mesenteric and retroperitoneal lymph nodes], various leukemias [lymphoid hematopoietic tissue], multiple myeloma [vertebrae / thorax / rib / skull and long bone], etc .;
  • Nerve fiber [systemic cutaneous nerve / deep nerve and internal organs], (malignant) schwannoma [nervous of head, neck, limbs, etc.], (malignant) glioblastoma [brain], medulloblastoma [ Cerebellum], (malignant) meningiomas [meninges], ganglioblastoma / neuroblastoma [mediastinum and retroperitoneum / adrenal medulla], etc .;
  • malignant melanoma [skin, mucosa], (malignant) hydatidiform mole, chorionic epithelial cancer [uterine], (malignant) supporter cells, stromal cell tumor, (malignant) granulosa cell tumor [ovarian, testicular], fine Protocytoma [testis], asexual cell tumor [ovary], embryonal cancer [testis, ovary], (malignant) teratoma [ovary, testis, mediastinum, and tail of the palate], etc .; 4. Regulate gene activity.
  • cAMP causes changes in enzyme and protein activity and ultimately changes in metabolic levels, and also regulates the activity of genes involved in the synthesis of certain enzymes.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of various diseases, such as diseases related to the dysfunction of the cell signaling system (such as the cAMP signaling pathway).
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) the cell signaling protein MyD24.09. Agonists increase the cellular signaling protein MyD24. 09 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 the cell signaling protein MyD24. 09 can be cultured with the labeled cell signaling protein MyD24. 09 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of the cell signaling protein MyD24.09 include screened antibodies, compounds, receptor deletions, and the like.
  • the antagonist of the cell signaling protein MyD24.09 can bind to the cell signaling protein MyD24.09 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 exert its biology Features.
  • the cell signaling protein MyD24.09 can be added to a bioanalytical assay to determine whether the compound is a compound by measuring the effect of the compound on the interaction between the cell signaling protein MyD24.09 and its receptor. Antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Peptide molecules capable of binding to the cell signaling protein MyD24.09 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the molecular signalling protein MyD24.09 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 cell signaling protein MyD24. 09 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting the cell signaling protein MyD24.09 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's Agent.
  • 'Techniques for preparing monoclonal antibodies to the cell signaling protein MyD24. 09 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta -Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using known techniques (Morri et al, PNAS, 1985, 81: 6851). And the unique technology for producing single chain antibodies (US Pat No. 4946778) Can also be used to produce single-chain antibodies against the cell signaling protein MyD24. C9.
  • An antibody against the cell signaling protein MyD24. 0 can be used in immunohistochemical techniques to detect the cell signaling protein MyD24. 09 in biopsy specimens.
  • Monoclonal antibodies that bind to the cell signaling protein MyD24. 09 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 against a specific bead site in the body.
  • the cell signaling protein MyD24. 09 high affinity monoclonal antibody can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol 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 the cell signaling protein MyD24. 09 positive cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to the cell signaling protein MyD24.09. Administration of an appropriate dose of antibody can stimulate or block the production or activity of the cell signaling protein MyD24.09.
  • the present invention also relates to a diagnostic test method for quantitatively and locally detecting the level of the cell signaling protein MyD24.09. These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of the cell signaling protein MyD24. 09 detected in the test can be used to explain the importance of the cell signaling protein MyD24. 09 in various diseases and to diagnose diseases where the cell signaling protein MyD24. 09 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.
  • the polynucleotide encoding the cell signaling protein MyD24.09 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 the cell signaling protein MyD24.09.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express the mutated cell signaling protein MyD24.09 to inhibit endogenous cell signaling protein MyD24.09 activity.
  • a mutated cellular signaling protein MyD24.09 may be a shortened cellular signaling protein MyD24.09 that lacks a signaling functional domain. Although it can bind to downstream substrates, it lacks signaling activity.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of the cell signaling protein MyD24.09.
  • Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. are available. They are used to transfer a polynucleotide encoding a cell signaling protein MyD24.09 into a cell. Constructing a Carrying Encoding Cell Methods for recombinant viral vectors of the polynucleotide MyD24.09 can be found in the literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding the cell signaling protein MyD24.09 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 MA and DNA
  • ribozymes that inhibit the cell signaling protein MyD24. 09 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target MA for endonucleation.
  • Antisense MA and DM and ribozymes can be obtained by any of the existing RNA or DNA synthesis techniques, such as the technique of solid phase phosphoramidite synthesis of oligonucleotides, which are widely used.
  • Antisense RM molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the NA. This DM sequence has been integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding the cell signaling protein MyD24. 09 can be used to diagnose diseases related to the cell signaling protein MyD24. 09.
  • the polynucleotide encoding the cell signaling protein MyD24. 09 can be used to detect the expression of the cell signaling protein MyD24. 09 or the abnormal expression of the cell signaling protein MyD24. 09 in a disease state.
  • the DM sequence encoding the cell signaling protein MyD24. 09 can be used to hybridize biopsy specimens to determine the expression status of the cell signaling protein MyD24.
  • Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These technical methods are all mature technologies that are publicly available, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also called a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • the transcription product of the cell signaling protein MyD24. 0 can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using the specific signaling protein MyD24. 09.
  • Detection of mutations in the cell signaling protein MyD24.09 gene can also be used to diagnose diseases related to the cell signaling protein MyD24.09.
  • Mutations of the cell signaling protein MyD24.09 include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type cell signaling protein MyD24.09 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for labeling chromosome positions.
  • an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared according to cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, 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 cDM libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopk ins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that are mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 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 that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • the cell signaling protein MyD24. 09 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of cell signaling protein MyDM. 09 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.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine MyD24.09 de transmission de signaux cellulaires, 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 de maladies associées aux dysfonctionnements du système de transmission de signaux cellulaires (à savoir le passage de signaux cAMP). L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine MyD24.09 de transmission de signaux cellulaires.
PCT/CN2001/001117 2000-07-07 2001-07-02 Nouveau polypeptide, proteine myd24.09 de transmission de signaux cellulaires, et polynucleotide codant ce polypeptide Ceased WO2002020777A1 (fr)

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US5955281A (en) * 1993-07-13 1999-09-21 Acadia Pharmaceuticals, Inc. Identification of ligands by selective amplification of cells transfected with receptors

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US5955281A (en) * 1993-07-13 1999-09-21 Acadia Pharmaceuticals, Inc. Identification of ligands by selective amplification of cells transfected with receptors

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DATABASE PROTEIN [online] 1 March 2000 (2000-03-01), SZENDRO P.T. ET AL., retrieved from GI:7110512 accession no. NCBI Database accession no. (AAF36973.1) *
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