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WO2002002614A1 - Nouveau polypeptide, proteine humaine 14 contenant un domaine de cytochrome c tnfr/ngfr, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 14 contenant un domaine de cytochrome c tnfr/ngfr, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002002614A1
WO2002002614A1 PCT/CN2001/000957 CN0100957W WO0202614A1 WO 2002002614 A1 WO2002002614 A1 WO 2002002614A1 CN 0100957 W CN0100957 W CN 0100957W WO 0202614 A1 WO0202614 A1 WO 0202614A1
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Prior art keywords
polypeptide
polynucleotide
domain
tnfr
protein
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PCT/CN2001/000957
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Application filed by Biowindow Gene Development Inc. Shanghai filed Critical Biowindow Gene Development Inc. Shanghai
Priority to AU95380/01A priority Critical patent/AU9538001A/en
Publication of WO2002002614A1 publication Critical patent/WO2002002614A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human TNFR / NGFR protein 14 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) ligase surface receptors; (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 heme group is tightly bound to two conserved cysteine residues through a thioether bond.
  • the binding site contains a consensus sequence: C-CPWHF-CPWR-C-H-CFYW; the histidine in the sequence is one of the two central ligands of the heme iron.
  • This conserved sequence is an important structural region where the protein binds to iron ions to form a specific structure to complete the process of electron transfer and energy conversion. Mutations in this central region are 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 novel 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 similar to other members of the family, it has similar biological functions. It is in vivo with various malignant tumors and cancers, various nerves Systemic disorders, related to various developmental disorders.
  • the analysis of the gene chip revealed that in the bladder mucosa, PMA + Ecv 304 cell line, LPS + Ecv 304 cell line thymus, normal fibroblasts 1024NC, F i brob last, growth factor stimulation, 1 024NT, scar into fc growth factor Stimulation, 1013HT, scar into fc to stimulate with growth factors, 101 3HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunum adenocarcinoma, cardia cancer
  • the expression profile of the polypeptide of the present invention is very similar to the expression profile of the human cytochrome C domain-containing TNFR / NGFR protein, so the functions of the two may also be similar.
  • the present invention has been named human TNFR / NGFR protein 14 containing a cytochrome C domain.
  • the human cytochrome C domain-containing TNFR / NGFR protein 14 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. More human cytochrome C domain-containing TNFR / NGFR protein 14 proteins need to be identified, especially the amino acid sequence of this protein. Isolation of the TKFR / NGFR protein 14 protein encoding 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, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human cytochrome C domain-containing TNFR / NGFR protein 14.
  • 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 / NGFR protein 14.
  • Another object of the present invention is to provide a method for producing a human cytochrome C domain-containing TNFR / NGFR protein 14.
  • Another object of the present invention is to provide an antibody against the human cytochrome C domain-containing TNFR / WGFR protein 14 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a human cytochrome c domain
  • TNFR / NGFR protein 14 mimics, antagonists, agonists, inhibitors.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human cytochrome C domain-containing TNFR / NGFR protein 14.
  • 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 279-662 in SEQ ID NO: 1; and (b) a sequence having 1-1771 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 method for screening compounds that mimic, activate, antagonize or inhibit the activity of human TNFR / NGFR protein 14 protein containing cytochrome C domain, which comprises using the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or disease susceptibility associated with abnormal expression of a human cytochrome C domain-containing TNFR / NGFR protein 14 protein, which comprises detecting the polypeptide or a susceptibility in a biological sample. A mutation in a coding polynucleotide 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 human cytochrome C domain-containing TNFR / NGFR protein 14. Use of medicine.
  • 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 / NGFR protein 14, 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 / NGFR protein 14.
  • An "antagonist” or “inhibitor” refers to an organism that can block or regulate human cytochrome C domain-containing TNFR / NGFR protein 14 when combined with human cytochrome C domain-containing TNFR / NGFR protein 14.
  • Molecularly active or immunologically active molecule Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human cytochrome C domain-containing TNFR / NGFR protein 14.
  • Regulation refers to changes in the function of human cytochrome C domain-containing TNFR / NGFR protein 14, including increased or decreased protein activity, changes in binding properties, and human cytochrome C domain-containing TNFR / NGFR protein 14 Of any other biological, functional or immune properties.
  • substantially pure ' means 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 using standard protein purification techniques.
  • / NGFR protein 14 Essentially pure human cytochrome C domain-containing TNFR / NGFR protein I 4 produces a single main band on a non-reducing polyacrylamide gel.
  • Human cytochrome C domain-containing TNFR / The purity of NGFR protein 14 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. This inhibition of hybridization can be detected by performing hybridization (Southern blotting or Nor thern 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 sof tware package, DNASTAR, Inc., Mad Son Wis.).
  • the MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higg ins, DG and PM Sharp (1988) Gene 73: 237-244).
  • the C Luster method sorts each group by checking the distance between all pairs. The columns are arranged in 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:
  • the number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the C l uster method or using methods known in the art such as; iotmi He in to determine the identity between nucleic acid sequences Percentage (Hein J., (1990) Methods in erazumo logy 183: 625-645) 0 "Similarity” refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RM sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “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, F (ab ') 2 and Fv, which can specifically bind to human cytochrome C domain-containing TNFR / NGFR protein 14 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 'but the same polynucleotide or polypeptide is separated from some or all of the coexisting substances 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 existing in the natural state.
  • isolated human cytochrome C domain-containing TNFR / NGFR protein 14 means that human cytochrome C domain-containing TNFR / NGFR protein 14 is substantially free of other proteins, lipids, Sugars or other substances.
  • cytochrome C domain-containing TNFR / NGFR protein 14 can purify using standard protein purification techniques.
  • Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human cytochrome C domain-containing TNFR / NGFR protein polypeptide can be analyzed by amino acid sequence analysis.
  • the present invention provides a new polypeptide, a human TNFR / NGFR protein 14 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.
  • polypeptides of the invention may be glycosylated, or they may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of human cytochrome C domain-containing TNFR / NGFR protein 14.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human cytochrome C domain-containing TNFR / NGFR protein 14 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: U) a type 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 substituted An amino acid may or may not be encoded by a genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (in) such a Species, wherein the mature polypeptide is fused to another compound (such as a compound that prolongs the polypeptide's half-life, such as polyethylene glycol); or (IV) such a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (such as Leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences) As set forth herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the 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 1771 bases in length and its open reading frames 279-662 encode 127 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile to human cytochrome C domain-containing TNFR / NGFR protein.
  • the human cytochrome C domain-containing TNFR / NGFR protein 14 has human cells.
  • the TNFR / NGFR protein of the pigment C domain functions similarly.
  • the polynucleotide of the present invention may be in the form of DM or RM.
  • DNA forms include cDNA, genomic DNA or artificially synthesized 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 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, 6 (TC; or (2) ) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1 ° /.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biology as the mature polypeptide shown in SEQ ID NO: 2 Function and activity.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human cytochrome C domain-containing TNFR / NGFR protein 14.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • Specific polynucleosides encoding human cytochrome C domain-containing TNFR / NGFR protein 14 of the present invention The acid sequence can be obtained in a variety of ways.
  • 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 CDM 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 DM 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.
  • Library is constructed cDM conventional method (Sambrook, et a l., Mo l ecular Cloning, A Laboratory Manua l, Co ld Spr ing Harbor Laboratory. New York, 1989) 0 commercially available cDNA library may also be obtained, such as C Different cDNA libraries from lontech. 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-DM or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human cytochrome C domain-containing TNFR / NGFR protein 14 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 / NGFR protein 14 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELI SA) and so on.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELI SA) and so on.
  • a method for amplifying DNA / RM using PCR technology is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and use regular Method synthesis.
  • 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, 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 cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a human cytochrome C domain-containing TNFR / NGFR protein 14 coding sequence. And a method for producing the polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding a human cytochrome C domain-containing TNFR / NGFR protein 14 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.
  • 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.
  • An expression vector of the DNA sequence of TNFR / NGFR protein 14 and appropriate transcriptional / translational regulatory elements include in vitro recombination MA technology, DM synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manua, cold Harbor Harbor Laboratory. New York, 1989).
  • the DM sequence can be operably linked to an appropriate promoter in an expression vector to guide mMA synthesis. Representative examples of these promoters are: l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pair SV40 enhancers on the late side of the origin of replication, and many SV40 enhancers on the late side of the origin of replication. Tumor enhancer and adenovirus enhancer.
  • 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 / NGFR protein 14 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a polynucleotide containing the polynucleotide or the recombinant vector.
  • Genetically engineered host cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • -Transformation of a host cell with a DM sequence or a recombinant vector containing the DNA sequence according to the present invention can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli, it can absorb
  • DNA competent cells harvested after exponential growth phase, treated with CaC l 2 method used in the step are 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 / NGFR protein 14 (Scence, 1984; 224: 1431). Generally there are the following steps:
  • 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 into a cell. Extracellular.
  • 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
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the human cytochrome C domain-containing TNFR / NGFR protein 14 and human cytochrome C domain-containing TNFR / NGFR protein.
  • the upper graph is a graph of the human cytochrome C domain-containing TNFR / NGFR protein 14.
  • the lower graph is the graph of the human cytochrome C domain-containing TNFR / NGFR protein.
  • 1-bladder mucosa 2-PMA + Ecv304 cell line, 3- LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC, 5-Fibroblas t, growth factor stimulation, 1024NT, 6-scars into fc growth factor Stimulation, 1013HT, 7-scar into fc without stimulation with growth factors, 1013HC, 8-bladder cancer cell EL 9-bladder cancer, 10-bladder cancer, 11-liver cancer, 12-liver cancer cell line, 13-fetal skin , 14-spleen, 15-prostate cancer, 16-jejunal adenocarcinoma, 17 cardia cancer.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human TNFR / NGFR protein 14 containing cytochrome C domain.
  • kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Example 1 Cloning of human cytochrome C domain-containing TNFR / NGFR protein 1.4
  • 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 Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA Cloning Kit purchased from Clontech was used to insert the CDM fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5 ⁇ to form a CDM library.
  • Dye terminate cycle react ion sequencing kit Perk in-Elmer
  • ABI 377 An automatic sequencer (Perkin-Elmer) determined the sequences at the 5 'and 3' ends of all clones. 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 0882ell was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the 0882ell clone contains a full-length cDNA of 1771bp (as shown in Seq ID NO: 1), and a 384bp open reading frame (0RF) from 279bp to 662bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-0882ell and the encoded protein was named human cytochrome C domain-containing TNFR / NGFR protein.
  • Example 2 RT-PCR method was used to clone the human cytochrome C domain-containing TNFR / NGFR protein.
  • I 4 gene was synthesized from fetal brain cells using total MA as a template and ol igo-dT as a primer for reverse transcription reaction to synthesize cDNA.
  • PCR amplification was performed with the following primers:
  • Pr imerl 5 NGACCCACCCTCTTAAGGGAACAA-3 (SEQ ID NO: 3)
  • Pr imer 2 5'- CCGGCTTCCCTAAAGAGAGCAGTA -3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer 2 is the 3'-end reverse sequence in SEQ ID NO: 1.
  • Conditions of the amplification reaction 50 mmol / L KC1, 10 ol / L Tris-Cl, (pH 8.5.5), 1.5 mmol / L MgCl 2 200 ⁇ mol / L dNTP in a reaction volume of 50 ⁇ 1 , lOpmol primer, 1U Taq DM 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 P CR vector (Invitrogen product) using a TA cloning kit.
  • DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 1771bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human cytochrome C domain-containing TNFR / NGFR protein 14 gene expression:
  • RNA extraction in one step [Anal. Biochera 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue was homogenized with 4M guanidinium isothiocyanate-25raM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA With 20 g of RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-lniM EDTA-2. 2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation of MA 32 P dATP labeled probe 32 ⁇ - by random priming Method - with oc.
  • the DM probe used is the human cytochrome C domain-containing TNFR / NGFR protein 14 coding region sequence (279bp to 662bp) amplified by PCR as shown in FIG.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KK pH7.4. 4 )-5 X SSC- 5 X Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in 1> ⁇ SSC-0.1% 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 human cytochrome C domain-containing TNFR / NGFR protein 14 Based on the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers was designed The sequence is as follows:
  • Pr imer 3 5'-CCCCATATGATGGAAAGGAGAAAGCCCGTTTCC-3 '(Seq ID No: 5)
  • Pr imer 4 5'-CCCGAATTCTTACACGCGTGTGCCACCACGCTC-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and EcoRI digestion sites, respectively, followed by the 5 'and 3' ends of the target gene, respectively.
  • the sequence, Ndel and EcoRI restriction sites correspond to selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3).
  • PCR was performed using the pBS-0882ell plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0882ell plasmid, primers Primer-3 and Primer-4 were 1 Opmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 4 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and EcoRI were used to double digest the amplified product and plasmid pET_28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed into Escherichia coli DH5 ct by the calcium chloride method.
  • cytochrome C domain-containing TNFR / NGFR protein was synthesized using a peptide synthesizer (product of PE) 14 specific peptides:
  • NH2-Met-Glu-Arg-Arg-Arg-Arg-Lys-Pro-Val-Ser-Lys-Arg-Ser-Pro-Arg-Pro-Phe-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 a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to human cytochrome C domain-containing TNFR / NGFR protein 14.
  • 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, Northern blotting, and copying methods. They all use the same steps to hybridize the fixed polynucleotide sample to 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 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 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.
  • 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, then 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 mutation sequence of the SBQ ID NO: 1 gene fragment or its complementary fragment (41Nt):
  • PBS phosphate buffered saline
  • phenol extraction method for DNA Steps 1) Wash the cells with 1-10 ml of cold PBS and centrifuge at 1,000 g for 10 minutes. 2) Resuspend the pelleted cells (1 x 10 8 cells / ml) with cold cell lysate and apply a minimum of 100 ⁇ l lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is directly added to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 7 cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) Incubate at 50 ° C for 1 hour or shake gently at 37 ° C overnight.
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each test #, so that they can be used in the subsequent experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • CT A1 (calf thymus DNA).
  • X-ray autoradiography -70 ° C
  • X-ray autoradiography press time depends on the radioactivity of the hybrid spot
  • Example 7 DM Microarray
  • Gene chip or gene micro-matrix 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.
  • the data is compared and analyzed on a carrier such as glass, silicon, and the like by fluorescence detection and computer software, so as to achieve the purpose of analyzing biological information quickly, efficiently, and with high throughput.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; searching for 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 various in the literature. Reports can be found in the literature DeRi s i, 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 respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500 ng / ul after purification, and spotted on a glass medium with 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 UV cross-linker. After elution, the slides were fixed to prepare DNA on a glass slide to prepare a chip. The specific method steps are widely reported in the literature. The post-spot processing steps in this embodiment are:
  • Total mRNA was extracted from the human mixed tissue and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified with Ol igotex mRNA Midi Ki t (purchased from QiaGen).
  • Cy3dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-tr iphate cou led to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech), a fluorescent reagent, was used to label mRM 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 the body's specific tissues (or stimulated cell lines) mRM, and the probes were prepared after purification.
  • Cy3dUTP (5-Amino-propargyl-2
  • Solut ion (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1> ⁇ SSC, 0.2% SDS) at room temperature and scanned with a ScanArray 3000 scanner (purchased from General Scanning, USA). The scanned image was analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibrob last, growth factor stimulation, 1024NT, scar-like fc growth factor Stimulation, 1013HT, scar into fc without stimulation with growth factors, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunum adenocarcinoma, cardia cancer. Draw a graph based on these 17 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the human cytochrome C domain-containing TNFR / NGFR protein 14 and the human cytochrome C domain-containing TNFR / NGFR protein expression profiles 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 [Ma l let S., Barclay AN, 1991, Immuno l 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 The combination of necrosis factor can effectively cause tumor cell response, promote tumor cell destruction, and inhibit infinite 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 nervous system 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 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 , Sex 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, Thymic tumors and nasal cavity.
  • Sinus tumors Sinus tumors, nasopharyngeal cancer, laryngeal cancer, tracheal tumors, pleural mesothelioma, fibroids, fibrosarcoma, lipoma, liposarcoma, leiomyoma, etc.
  • transient ischemic attack glioblastoma, meningiomas, neurofibro pituitary adenoma, intracranial granuloma, Alzheimer's disease, Parkinson's disease, chorea, depression, amnesia , Jonton's disease, epilepsy, migraine, dementia, multiple sclerosis, myasthenia gravis, spinal muscular atrophy, pseudohypertrophy, Duchenne muscular dystrophy, tonic muscular dystrophy, tonicity, retarded exercise Disorders Dystonia, Neurofibromatosis, Nodular Sclerosis, Cerebral Trigeminal Hemangioma, Ataxia.
  • Vasodilation Schizophrenia, Depression, Paranoia, Anxiety, Obsessive-Compulsive Disorder, Phobia , Neurasthenia 3 ⁇ 4 Acute myelitis, Spinal cord compression, Trigeminal neuralgia, Facial palsy, Rostral palsy, Sciatica, Liming-Barre syndrome, Neural tube insufficiency, Cerebral malformation, Down syndrome, Spinal deformity Sexual hydrocephalus.
  • These diseases include but are not limited to the following, such as: cleft palate, facial cleft palate, cervical sac, cervical fistula, limb absentness, limb differentiation disorders, digestive tract atresia or stenosis, ileal diverticulum, umbilical diaphragm, 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 Pee Fistula, crypto, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias, hermaphroditism, atrial septal defect, ventricular septal defect, abnormal arterial stem separation, aortic or
  • Abnormal expression of the human cytochrome C domain-containing TNFR / NGFR protein of the present invention will also produce certain genetic, hematological and immune system diseases.
  • the present invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human cytochrome C domain-containing TNFR / NGFR protein 14.
  • Agonists increase human cytochrome C domain-containing TNFR / NGFR protein 14 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human cytochrome C domain-containing TNFR / NGFR protein 14 can be cultured in the presence of drugs with labeled human cytochrome C domain-containing TNFR / NGFR protein 14. The ability of the drug to increase or suppress this interaction is then determined.
  • Antagonists of human cytochrome C domain-containing TNFR / NGFR protein 14 include selected antibodies, compounds, receptor deletions, and the like. Antagonist of human cytochrome C domain-containing TNFR / NGFR protein 14 can bind to human cytochrome C domain-containing TNFR / NGFR protein 14 and eliminate its function, or inhibit the production of the polypeptide, or with the polypeptide The active site binding prevents the polypeptide from performing biological functions.
  • human cytochrome C domain-containing TNFR / NGFR protein 14 can be added to the bioanalytical assay. 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 / NGFR protein 14 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, human cytochrome C domain-containing TNFR / NGFR protein 14 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 invention also provides a needle Antibody to human cytochrome C domain-containing TNFR / NGFR protein 14 epitope.
  • These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human cytochrome C domain-containing TNFR / NGFR protein 14 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 preparing human cytochrome C domain-containing TNFR / NGFR protein 14 monoclonal antibodies include, but are not limited to, hybridoma technology (Koliler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, Human B-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 (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies U.S. Pat No. 4946778, can also be used to produce single chain antibodies against human TNFR / NGFR protein 14 containing cytochrome C domain.
  • Antibodies against human cytochrome C domain-containing TNFR / NGFR protein 14 can be used in immunohistochemical techniques to detect human cytochrome C domain-containing TNFR / NGFR protein 14 in biopsy specimens.
  • Monoclonal antibodies that bind to human cytochrome C domain-containing TNFR / NGFR protein 14 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 / NGFR protein 14 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 / NGFR protein 14 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human cytochrome C domain-containing TNFR / NGFR protein 14.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human TNFR / NGFR protein 14 containing a cytochrome C domain.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human cytochrome C domain-containing TNFR / NGFR protein 14 levels.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the human cytochrome C domain-containing TNFR / NGFR protein 14 levels detected in the test can be used to explain the importance of human cytochrome C domain-containing TNFR / NGFR protein 14 in various diseases and to diagnose humans. Diseases in which the cytochrome C domain-containing TNFR / NGFR protein 14 functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human cytochrome C domain-containing TNFR / NGFR proteins 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 cytochrome C domain-containing TNFR / NGFR protein 14.
  • Recombinant gene therapy vectors can be designed to express mutated human cytochrome C domain-containing TNFR / NGFR protein 14 to inhibit endogenous human cytochrome C domain-containing TNFR / NGFR protein 14 active.
  • a variant human cytochrome C domain-containing TNFR / NGFR protein 14 may be a shortened human cytochrome C domain-containing TNFR / NGFR protein 14 without a signaling domain, although it may be related to downstream Substrate binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human cytochrome C domain-containing TNFR / NGFR protein 14.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human cytochrome C domain-containing TNFR / NGFR protein 14 to cells Inside.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human cytochrome C domain-containing TNFR / NGFR protein 14 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human cytochrome C domain-containing TNFR / NGFR protein 14 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human TNFR / NGFR protein 14 mRNA containing a cytochrome C domain are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RM, DM and ribozymes can be obtained by any of the existing RNA or DM synthesis techniques, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis, which is widely used.
  • Antisense MA molecules can be obtained by in vitro or in vivo transcription of DM sequences encoding the RNA. This DNA sequence is integrated downstream of the vector's RNA polymerase promoter. 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 / NGFR protein 14 can be used for diagnosis of diseases related to human cytochrome C domain-containing TNFR / NGFR protein 14.
  • the polynucleotide encoding human cytochrome C domain-containing TNFR / NGFR protein 14 can be used to detect the expression of human cytochrome C domain-containing TNFR / NGFR protein 14 or human cytochrome C domain-containing disease states Abnormal expression of TNFR / NGFR protein 14.
  • White 14's MA sequence can be used to hybridize biopsy specimens to determine the expression of human cytochrome C domain-containing TNFR / NGFR protein 14.
  • Hybridization techniques include Southern blotting, Nor thern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in a tissue.
  • Human cytochrome C domain-containing TNFR / NGFR protein 14 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human cytochrome C domain-containing TNFR / NGFR protein 14 transcripts .
  • Detection of mutations in the human cytochrome C domain-containing TNFR / NGFR protein 14 gene can also be used to diagnose human cytochrome C domain-containing TNFR / NGFR protein 14-related diseases.
  • Human cytochrome C domain-containing TNFR / NGFR protein 14 mutant forms include point mutations, translocations, deletions, recombinations, and others compared to normal wild-type human cytochrome C domain-containing TNFR / NGFR protein 14 DNA sequences Any exceptions etc. Mutations can be detected using well-known techniques such as Southern blotting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern blotting method and Wes tern imprinting method 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 DM 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 DM 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 To correlate with genetic map data. These data can be found, for example, in V. Mckusick, Mende Liaii Inheritance in Man (available online with Johns Hopkins University Wetch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping technology, the CDM that is 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 / NGFR protein 14 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 / NGFR protein 14 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 humaine 14 contenant un domaine de cytochrome c TNFR/NGFR, 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 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 la protéine humaine 14 contenant un domaine de cytochrome c TNFR/NGFR.
PCT/CN2001/000957 2000-06-14 2001-06-11 Nouveau polypeptide, proteine humaine 14 contenant un domaine de cytochrome c tnfr/ngfr, et polynucleotide codant ce polypeptide WO2002002614A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU95380/01A AU9538001A (en) 2000-06-14 2001-06-11 A novel polypeptide - human cytochrome c domain containing-tnfr/ngfr protein 14 and a polynucleotide encoding the same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN 00116496 CN1328051A (zh) 2000-06-14 2000-06-14 一种新的多肽——人含细胞色素c结构域的tnfr/ngfr蛋白14和编码这种多肽的多核苷酸
CN00116496.1 2000-06-14

Publications (1)

Publication Number Publication Date
WO2002002614A1 true WO2002002614A1 (fr) 2002-01-10

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PCT/CN2001/000957 WO2002002614A1 (fr) 2000-06-14 2001-06-11 Nouveau polypeptide, proteine humaine 14 contenant un domaine de cytochrome c tnfr/ngfr, et polynucleotide codant ce polypeptide

Country Status (3)

Country Link
CN (1) CN1328051A (fr)
AU (1) AU9538001A (fr)
WO (1) WO2002002614A1 (fr)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM., vol. 260, no. 13, 1985, pages 8044 - 8049 *
PROC. NATL. ACAD. SCI. USA, vol. 83, no. 5, 1986, pages 1403 - 1407 *

Also Published As

Publication number Publication date
AU9538001A (en) 2002-01-14
CN1328051A (zh) 2001-12-26

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