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WO2001074994A2 - Nouveau polypeptide, proteine tnfr/nger 13 contenant un domaine de cytochrome c, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine tnfr/nger 13 contenant un domaine de cytochrome c, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001074994A2
WO2001074994A2 PCT/CN2001/000220 CN0100220W WO0174994A2 WO 2001074994 A2 WO2001074994 A2 WO 2001074994A2 CN 0100220 W CN0100220 W CN 0100220W WO 0174994 A2 WO0174994 A2 WO 0174994A2
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Prior art keywords
polypeptide
polynucleotide
domain
protein
tnfr
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Chinese (zh)
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WO2001074994A3 (fr
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 AU44054/01A priority Critical patent/AU4405401A/en
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Publication of WO2001074994A3 publication Critical patent/WO2001074994A3/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
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/80—Cytochromes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide __human TNFR / NGER protein 13 containing a cytochrome C domain, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Hydrophilic signal molecules include neurotransmitters, growth factors, cytokines, local chemical transmitters, and most hormones. They cannot pass through the plasma membrane and can only bind to receptors on the cell surface to form ligand-receptor complexes for signaling. divert. According to the mechanism of signal transduction and the types of receptor proteins, cell surface receptors can be divided into three types: (1) ion channel-coupled receptors; (2) surface receptors for ligases; (3) with G proteins Coupling receptor.
  • nerve growth factor receptor proteins there are a large number of growth factor receptor proteins in the body, and these growth factor receptor proteins combine with various signal factors to correctly regulate various physiological responses in the body.
  • the nerve growth factor receptor and tumor necrosis factor receptor constitute an independent nerve growth factor receptor and tumor necrosis factor receptor (TNFR / NGFR) superfamily [Mallet S., Barclay AN, 1991, Immunol Today, 15: 220-223].
  • Nerve growth factor receptor proteins may regulate the signal response of nerve cells to nerve growth factors in the body to coordinate the metabolism of the nervous system and exert normal physiological functions; and tumor necrosis factor receptors in the body and its related signal factors such as tumors
  • necrosis factor can effectively cause tumor cell response, promote tumor cell destruction, and inhibit infinite proliferation of tumor cells.
  • the abnormal expression of these proteins will lead to abnormal growth and development of the nervous system, abnormal proliferation of tissue cells and abnormal expression of proteins, which will cause various related diseases, such as various malignant tumors and cancers, and various nervous systems. Illness, various developmental disorders, etc.
  • cysteine-rich domain consisting of 110-160 amino acid residues.
  • This domain contains the following conserved consensus sequence fragments: CX (4, 6)-[FYH]-X (5, 10)-CX (0, 2)-CX (2, 3)-CX (7, 11) -CX (4, 6)-[DNEQSKP]-X (2)-C.
  • This domain can be divided into four different patterns.
  • the binding site contains a consensus sequence:
  • C- ⁇ CPWHF ⁇ - ⁇ CPWR ⁇ -C-H- ⁇ CFYW ⁇ ; Histidine in the sequence is one of the two central ligands of heme iron.
  • This conserved sequence is an important structural region where proteins bind to iron ions to form a specific structure to complete the process of electron transfer and energy conversion.
  • the mutation in this central region is likely to be the direct cause of the abnormal function of the protein, causing the protein to fail to complete various energy conversion processes normally, thereby causing various related diseases.
  • the new human cytochrome C domain-containing TNFR / NGFR protein of the present invention also contains N-terminally conserved amino acid sequence fragments of the nerve growth factor receptor and tumor necrosis factor receptor superfamily, and a conserved cytochrome C-conserved sequence fragment. Therefore, it is a new member of the human nerve growth factor receptor and tumor necrosis factor receptor superfamily, and has similar biological functions as other members of the family. It has similar biological functions in vivo with various malignant tumors and cancers, and various nerves. Systemic disorders, related to various developmental disorders.
  • the expression profile of the polypeptide of the present invention is very similar to the expression profile of human cytochrome C domain-containing TNFR / NGER protein 51, so the functions of the two may also be similar.
  • the invention is named human TNFR / NGER protein containing cytochrome C domain.
  • the human cytochrome C domain-containing TNFR / NGER protein 1 3 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes. There has been a need to identify more human cytochrome C domain-containing TNFR / NGER protein 13 proteins involved in these processes, especially the amino acid sequence of this protein. Isolation of the TNFR / NGER protein 1 3 gene containing cytochrome C domain in newcomers 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 and therefore it is important to isolate its coding for DM. Disclosure of invention
  • TNFR / NGER protein 1 3 and fragments, analogs and derivatives thereof.
  • 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 human cytochrome C domain-containing TNFR / NGER protein 13.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a human cytochrome C domain-containing TNFR / NGER protein 13.
  • Another object of the present invention is to provide human cytochrome C domain-containing TNFR / NGER protein.
  • Another object of the present invention is to provide an antibody directed against a human cytochrome C domain-containing TNFR / NGER protein 13 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of the TNFR / NGER protein 13 containing cytochrome C domain of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human cytochrome C domain-containing TNFR / NGER protein 13.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 2, or a conservative variant, biologically active fragment, or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID D0: 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 31 1 to 667 in SEQ ID NO: 1; and (b) a sequence having 1-1 in SEQ ID NO: 1 2452-bit sequence.
  • 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 present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human cytochrome C domain-containing TNFR / NGER protein 13 protein activity, which comprises utilizing the polypeptide of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of a human cytochrome C domain-containing TNFR / NGER protein 1 3 protein in vitro, comprising detecting the polypeptide or a polynucleotide encoding the same in a biological sample. A mutation in a sequence, or the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • the invention also relates to a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the preparation of polypeptides and / or polynucleotides of the present invention for the treatment of cancer, developmental or immune diseases, or other diseases caused by abnormal expression of TNFR / NGER protein 1 3 containing human cytochrome C domain. Use of drugs.
  • 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 DNA 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 and to bind to specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human cytochrome C domain-containing TNFR / NGER protein 1 3, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to a human cytochrome C domain-containing TNFR / NGER protein 1 3.
  • Antagonist refers to TNFR / NGER protein when it is associated with human cytochrome C domain 1 3 When bound, a molecule that can block or regulate the biological or immunological activity of human cytochrome C domain-containing TNFR / NGER protein 13.
  • Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human cytochrome C domain-containing TNFR / NGER protein 13.
  • 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 human cytochrome C domain-containing TNFR / NGER protein 13 using standard protein purification techniques.
  • the substantially pure human cytochrome C domain-containing TNFR / NGER protein 13 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of human cytochrome C domain-containing TNFR / NGER protein 13 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
  • 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. The inhibition of such hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of a completely homologous sequence to a 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 interact with each other specifically or selectively.
  • 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 (Lasergene software package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Cluster method arranges each group of sequences by checking the distance between all pairs. Into clusters. 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:
  • nucleic acid sequences 100 Number of residues in sequence A-number of spaced residues in sequence A-number of spaced residues in sequence B
  • percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in emzumology 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and 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” means
  • Sense strand A complementary nucleic acid strand.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, 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, F (ab ') 2 and Fv, which can specifically bind to human cytochrome C domain-containing TNFR / NGER protein 13 epitopes.
  • 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 matter 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 vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not a component 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 human cytochrome C domain-containing TNFR / NGER protein 13 means that human cytochrome C domain-containing TNFR / NGER protein 13 is substantially free of other proteins, lipids, Sugars or other substances. Those skilled in the art can purify human cytochrome C domain-containing TNFR / NGER protein 13 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human cytochrome C domain-containing TNFR / NGER protein 13 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide—a human TNFR / NGER protein 13 containing a cytochrome C domain, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human cytochrome C domain-containing TNFR / NGER protein 13.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human cytochrome C domain-containing TNFR / NGER protein 13 of the present invention. .
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by the genetic code; or (II) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a 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 the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • such fragments, 00 derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 2452 bases in length and its open reading frames 311-667 encode 118 amino acids.
  • this polypeptide has a similar expression profile to human cytochrome C domain-containing TNFR / NGER protein 51, and it can be inferred that the human cytochrome C domain-containing TNFR / NGER protein 1 3 has human Cytochrome C domain-containing TNFR / NGER protein 51 functions similarly.
  • the polynucleotide of the present invention may be in the form of DM or RNA.
  • the DNA form includes cDM, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide 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 present invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only in two sequences Crosses occur only when the identity between them is at least 95%, and more preferably 97%.
  • 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, most preferably at least 100 More than nucleotides.
  • Nucleic acid fragments can also be used in nucleic acid amplification techniques (eg, PCR) to identify and / or isolate polynucleotides encoding human cytochrome C domain-containing TNFR / NGER protein 1 3.
  • 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 human cytochrome C domain-containing TNFR / NGER protein 13 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene).
  • cDNA libraries are also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human cytochrome C domain-containing TNFR / NGER protein 13 The level of transcripts; (4) Detecting protein products expressed by genes by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is 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 of human cytochrome C domain-containing TNFR / NGER protein 13 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.
  • the RACE method RACE-rapid cDNA end rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein.
  • 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 cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length CDM sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human cytochrome C domain-containing TNFR / NGER protein 13 coding sequence, and produced by recombinant technology A method of a polypeptide according to the invention.
  • a polynucleotide sequence encoding a human cytochrome C domain-containing TNFR / NGER protein 13 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, 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. Gene, 1987,
  • pMSXND expression vectors (Lee and Nathans, J Bio Chem. 263: 3521, 1988) expressed in mammalian cells and baculovirus-derived vectors expressed in insect cells.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain replication origins, promoters, marker genes, and translational regulatory elements.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • promoters are: the lac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, and the early and late SV40 promoters , Retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation.
  • Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription.
  • Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a human cytochrome C domain-containing TNFR / NGER protein 13 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute the polynucleotide or the recombinant vector. Genetically engineered host cells.
  • 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.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf 9; animal cells such as CH0, COS, or Bowes s melanoma cells, etc. .
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with CaC l 2 method used in steps well known in the art. The alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human cytochrome C domain-containing TNFR / NGER protein 1 (Scence, 1 984; 224: 14 31). Generally there are the following steps:
  • the polynucleotide (or variant) encoding the human-human cytochrome C domain-containing TNFR / NGER protein 13 or the recombinant expression vector containing the polynucleotide is suitable for transformation or transduction.
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • 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 chromat
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human cytochrome C domain-containing TNFR / NGER protein 13 and human cytochrome C domain-containing TNFR / NGER protein 51.
  • FIG. The upper graph is a graph of the expression profile of human cytochrome C domain-containing TNFR / NGER protein 13 and the lower sequence is the graph of the expression profile of human cytochrome C domain-containing TNFR / NGER protein 51.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human TNFR / NGER protein 13 containing cytochrome C domain. 13kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • Example 1 Cloning of human cytochrome C domain-containing TNFR / NGER protein 13
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. The 00 ⁇ fragment was directionally inserted into a multicloning site of a pBSK (+) vector (product of Clontech) using a Smart cDNA cloning kit (purchased from C1 ontech) to transform DH5 ⁇ , and the bacteria formed a cDNA library.
  • Dye terminate cycle reaction ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0104cll was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the 0104cll clone contained a full-length cDNA of 2452bp (as shown in Seq ID NO: 1), and a 357bp open reading frame (0RF) from 311bp to 667bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-0104cl 1 was named human cytochrome C domain-containing TNFR / NGER protein 13.
  • Example 2 Cloning of a gene encoding human cytochrome C domain-containing TNFR / NGER protein 13 by RT-PCR CDNA was synthesized using fetal brain cell total RNA 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:
  • Primer2 5,-ACAGAGTCTCGCTTTGTTGCCCAG -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KC1, 10 mmol / L in a 50 ⁇ l reaction volume
  • Tris-Cl (pH8.5), 1.5mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector using a TA cloning kit (Invitrogen).
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidinium 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) are added. ) And centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH 7. 4)-5 X SSC- 5 X Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, place the filter at 1 x
  • Example 4 In vitro expression, isolation and purification of recombinant human cytochrome C domain-containing TNFR / NGER protein 13
  • a pair of specific amplification primers were designed based on the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1 The sequence is as follows: Primer3: 5'-CCCCATATGATGACTGGTATAACCCAAGTGAAA-3 '(Seq ID No: 5) Primer4: 5,-CCCGAATTCTTAACTGCAGACATCCAGCCTTTG- 3, (Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and BaraHI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • the PCR reaction was performed using the pBS-0104cll plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-0104cll-containing plasmid in a total volume of 50 ⁇ l, primers Primer-3 and Primer-4 were lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94.
  • 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 ligated product was transformed into E. coli DH5cc using the calcium chloride method. After being cultured overnight in LB plates containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (PET-0104cll) with the correct sequence was selected, and the recombinant plasmid was transformed into E.
  • coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • the host strain BL21 (pET-0104cll) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 ol / L.
  • IPTG was added to a final concentration of 1 ol / L.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation.
  • a purified human cytochrome C domain-containing TNFR / NGER protein 13 was purified. After SDS-PAGE electrophoresis, a single band was obtained at 13 kDa ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2.
  • NH2-Met-Thr-Gly-Ile-Thr-Gln-Val-Lys-Asn-Asn-Ser-Gly-Leu-Asp-Glu-C00 H (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. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with 15 ⁇ g / ml bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Seph a rose 4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • Immunoprecipitation demonstrated that purified antibodies can specifically bind to human cytochrome C domain-containing TNFR / NGER protein 13 binds.
  • 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 example 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.
  • Filter hybridization methods include dot blotting, Southern blotting, Nor thern blotting, and copying methods, etc. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps.
  • 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 the labeled probe and incubated to hybridize the probe 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) to reduce the hybridization background and retain only strong specific signals.
  • 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 spot imprint 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
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • 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, then the primary probe should not be used; 5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.
  • Probe l 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 (probe2), 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
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • the hybridization experiments performed under low-intensity membrane washing conditions did not differ significantly in the radioactivity of the hybridization spots of the above two probes; while the hybridization experiments conducted under high-intensity membrane washing conditions, the radioactive intensity of hybridization spots of probe 1 was significantly stronger than The radioactive intensity of the hybridization spot of the other probe. Therefore, the presence and differential expression of the polynucleotide of the present invention in different tissues can be analyzed qualitatively and quantitatively with the probe 1.
  • Gene microarray or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, silicon Wait on the carrier, then use fluorescence detection and computer software for data comparison and 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 a target DM for gene chip technology for high-throughput research on the function of new genes; finding and screening new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, see DeRisi, J. L., Lyer, V. & Brown, P.0.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotting instrument (purchased from Cartesian, USA). The distance is 280 ⁇ ! . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DM on the glass slides to prepare chips. The specific method steps have been variously reported in the literature. The post-spot processing steps of this embodiment are:
  • the probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (lx SSC, 0.2% SDS) at room temperature and scanned with ScanArray 3000.
  • the instrument purchased from General Scanning Company, USA
  • the scanned image was analyzed and processed with Imagene software (Biodiscovery Company, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Plot a graph based on these 13 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profile of human cytochrome C domain-containing TNFR / NGER protein 13 and human cytochrome C domain-containing TNFR / NGER protein 51 are very similar. Industrial applicability
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Nerve growth factor receptor and tumor necrosis factor receptor constitute an independent nerve growth factor receptor and tumor necrosis factor receptor (TNFR / NGFR) superfamily [Mallet S., Barclay AN, 1991, I Painting unol Today, 15: 220-223] 0 Nerve growth factor receptor protein may regulate the signal response of nerve cells to nerve growth factor in the body to coordinate the metabolism of the nervous system and exert normal physiological functions; and tumor necrosis factor Receptor binding in vivo with its related signal factors such as tumor necrosis factor can effectively cause tumor cell response, promote tumor cell destruction, and inhibit unlimited proliferation of tumor cells.
  • the cysteine-rich domain of this specific TNFR / NGFR family is the central region of the active conformation of the receptor protein, and plays an important regulatory role in the normal function of the protein. Abnormal expression of such proteins will lead to the nervous system's Abnormal growth and development, as well as abnormal proliferation of tissue cells and abnormal expression of proteins, cause a variety of related diseases, such as: various malignant tumors and cancers, various nervous system disorders, and various development disorders.
  • the abnormal expression of the human cytochrome C domain-containing TNFR / NGFR protein of the present invention will Produce various diseases, especially various tumors, various nervous system disorders, various development disorders, etc. These diseases include, but are not limited to:
  • tumors gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, colon cancer , Malignant histiocytosis, melanoma, teratoma, sarcoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, Tumors of the thymus and nasal sinuses, nasopharyngeal carcinoma, laryngeal carcinoma, tracheal tumors, pleural mesothelioma, fibroids, fibrosarcoma, lipoma, liposarcoma, leiomyoma, etc.
  • transient ischemic attack glioblastoma, meningiomas, neurofibromas, pituitary adenomas, intracranial granulomas, dementia, Parkinson's disease, chorea, depression, Amnesia, Huntington's disease, epilepsy, migraine, dementia, multiple sclerosis, myasthenia gravis, spinal muscular atrophy, muscular pseudohypertrophy, Duchenne muscular dystrophy, tonic muscular dystrophy, myotonic , Bradykinesia, dystonia, neurofibromatosis, nodular sclerosis, trigeminal neurohemangioma, ataxia telangiectasia, schizophrenia, depression, paranoia, anxiety, obsessive-compulsive disorder , Phobia, neurodegeneration, acute myelitis, spinal cord compression, trigeminal neuralgia, facial nerve palsy, bulbar palsy, sciatica, Guillain-Barre syndrome, neural tube insufficiency, brain
  • These diseases include, but are not limited to, the following, such as: cleft palate, facial cleft lip, cervical sac, cervical fistula, lack of limbs, limb differentiation disorders, obstruction or stenosis of the digestive tract, ileal diverticulum, umbilical fistula, congenital umbilical Hernia, congenital aganglion-free giant colon, laryngotracheal stenosis or atresia, tracheoesophageal fistula, hyaline membrane disease, congenital pulmonary cyst, atelectasis, polycystic kidney, ectopic kidney, horse telluride, double ureter, umbilical Urinary fistula, crypto, congenital abdominal hernia, double uterus, vaginal atresia, hypospadias, hermaphroditism, atrial septal defect, ventricular septal defect, abnormal arterial stem separation, aortic or pulmonary
  • Danbol t-Cl os s syndrome Congenital lens position abnormality, Congenital blepharoplasia, Retinal dysplasia, Congenital optic nerve atrophy, Congenital sensorineural hearing loss, Hand cleft palate, Teratology, Wi lli ams syndrome, Al ag il le syndrome, Bayer syndrome, etc.
  • the abnormal expression of the human cytochrome C domain-containing TNFR / NGFR protein of the present invention will also generate certain hereditary, hematological and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human cytochrome C domain-containing TNFR / NGER protein 13. Agonists enhance human cytochrome C domain-containing TNFR / NGER protein 13 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human cytochrome C domain-containing TNFR / NGER protein 13 and a labeled human cytochrome C domain-containing TNFR / NGER protein 13 can be cultured in the presence of a drug. . The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human cytochrome C domain-containing TNFR / NGER protein 13 include selected antibodies, compounds, receptor deletions, and the like. Antagonist of human cytochrome C domain-containing TNFR / NGER protein 13 can bind to human cytochrome C domain-containing TNFR / NGER protein 13 and eliminate its function, or inhibit the production of the polypeptide, or The active site binding of a polypeptide prevents the polypeptide from performing its biological function.
  • human cytochrome C domain-containing TNFR / NGER protein 13 can be added to a bioanalytical assay, and the compounds can be used to determine human cytochrome C domain-containing TNFR / NGER protein 13 and its effects. The effects of interactions between humans to determine whether a compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human cytochrome C domain-containing TNFR / NGER protein 13 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, human cytochrome C domain-containing TNFR / NGER protein 13 molecules should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the present invention also provides antibodies against human cytochrome C domain-containing TNFR / NGER protein 13 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human cytochrome C domain-containing TNFR / NGER protein 13 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 it is not limited to Freund's adjuvant.
  • Techniques for making human cytochrome C domain-containing monoclonal antibodies to TNFR / NGER protein 13 include, but are not limited to, hybridoma technology (Kohler and Mistein.
  • Antibodies against human cytochrome C domain-containing TNFR / NGER protein 1 3 can be used in immunohistochemistry to detect human cytochrome C domain-containing TNFR / NGER protein 1 3 in biopsy specimens.
  • Monoclonal antibodies that bind to human cytochrome C domain-containing TNFR / NGER protein 1 3 can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human cytochrome C domain-containing TNFR / NGER protein 1 3 High affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human cytochrome C domain-containing TNFR / NGER protein 1 3 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human cytochrome C domain-containing TNFR / NGER protein 13.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human TNFR / NGER protein 13 containing a cytochrome C domain.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting human cytochrome C domain-containing TNFR / NGER protein 1 3 levels.
  • These tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the levels of human cytochrome C domain-containing TNFR / NGER protein 1 3 detected in the test can be used to explain the importance of human cytochrome C domain-containing TNFR / NGER protein 1 3 in various diseases and for Diagnosis of diseases in which human cytochrome C domain-containing TNFR / NGER protein 1 3 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.
  • Polynucleotides encoding human cytochrome C domain-containing TNFR / NGER protein 1 3 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human TNFR / NGER protein 1 containing cytochrome C domain.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human cytochrome C domain-containing
  • TNFR / NGER protein 1 3 to inhibit endogenous human cytochrome C domain-containing TNFR / NGER protein 13 activity.
  • a variant human cytochrome C domain-containing TNFR / NGER protein 1 3 may be a shortened human cytochrome C domain-containing TNFR / NGER protein 1 3, although it may be related to The downstream substrate binds but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of TNFR / NGER protein 13 containing cytochrome C domain.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a recombinant viral vector carrying a polynucleotide encoding a human cytochrome C domain-containing TNFR / NGER protein 13 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human cytochrome C domain-containing TNFR / NGER protein 13 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, etc.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides that inhibit human TNFR / NGER protein 13 mRNA containing a cytochrome C domain 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 RNA for endonuclease action.
  • Antisense RNA, DM and ribozymes can be obtained by any existing RNA or DNA synthesis technology. For example, the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RNA 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 phosphorothioate or peptide bond is used instead of the phosphodiester bond to link the ribonucleosides.
  • the polynucleotide encoding human cytochrome C domain-containing TNFR / NGER protein 13 can be used for diagnosis of diseases related to human cytochrome C domain-containing TNFR / NGER protein 13.
  • the polynucleotide encoding human cytochrome C domain-containing TNFR / NGER protein 13 can be used to detect the expression of human cytochrome C domain-containing TNFR / NGER protein 13 or human cytochrome C domain-containing disease states of
  • TNFR / NGER protein 13 Abnormal expression of TNFR / NGER protein 13.
  • the DNA sequence encoding human cytochrome C domain-containing TNFR / NGER protein 13 can be used to hybridize biopsy specimens to determine the expression of human cytochrome C domain-containing TNFR / NGER protein 13.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available. Part or all of the polynucleotides of the present invention can be immobilized on a microarray as probes
  • cytochrome C domain-containing TNFR / NGER protein 13 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human cytochrome C domain-containing TNFR / NGER protein 13 transcripts .
  • Detection of mutations in the human cytochrome C domain-containing TNFR / NGER protein 13 gene can also be used to diagnose human cytochrome C domain-containing TNFR / NGER protein 13-related diseases.
  • Mutated forms of human cytochrome C domain-containing TNFR / NGER protein 13 include TNFR / NGER protein 13 DNA sequence compared to point mutations, translocations, deletions, recombination and any other abnormalities. Mutations can be detected using existing techniques such as Southern blotting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern 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 marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be 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 pre-selection of hybridization to construct chromosome-specific cDNA 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, Mendel ian
  • the differences in cDNA or genomic sequences between the affected and unaffected individuals need 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 the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using 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.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them.
  • the polypeptide of the present invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human cytochrome C domain-containing TNFR / NGER protein 13 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human cytochrome C domain-containing TNFR / NGER protein 1 3 to be 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 TNFR/NGER 13 contenant un domaine de cytochrome c, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine TNFR/NGER 13 contenant un domaine de cytochrome c.
PCT/CN2001/000220 2000-03-02 2001-02-26 Nouveau polypeptide, proteine tnfr/nger 13 contenant un domaine de cytochrome c, et polynucleotide codant pour ce polypeptide Ceased WO2001074994A2 (fr)

Priority Applications (1)

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AU44054/01A AU4405401A (en) 2000-03-02 2001-02-26 A novel polypeptide, tnfr/nger protein 13 containing a cytochrome c domain and the polynucleotide encoding the polypeptide

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CN00111879.X 2000-03-02
CN 00111879 CN1311254A (zh) 2000-03-02 2000-03-02 一种新的多肽——人含细胞色素c结构域的 tnfr/nger蛋白13和编码这种多肽的多核苷酸

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IL120485A0 (en) * 1997-03-19 1997-07-13 Yeda Res & Dev Modulators of the function of receptors of the TNF/NGF receptor family and other proteins

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