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WO2001055381A1 - Nouveau polypeptide, facteur regulateur 80 de la transcription humaine, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, facteur regulateur 80 de la transcription humaine, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001055381A1
WO2001055381A1 PCT/CN2001/000082 CN0100082W WO0155381A1 WO 2001055381 A1 WO2001055381 A1 WO 2001055381A1 CN 0100082 W CN0100082 W CN 0100082W WO 0155381 A1 WO0155381 A1 WO 0155381A1
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Prior art keywords
polypeptide
polynucleotide
sequence
human
seq
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai BioDoor Gene Technology Ltd
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Shanghai BioDoor Gene Technology Ltd
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Priority to AU33561/01A priority Critical patent/AU3356101A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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, a human transcriptional regulatory factor 80, and a polynucleotide sequence encoding the polypeptide. The invention also relates to the preparation method and application of the polynucleotide and polypeptide. Background technique
  • Transcriptional regulators usually regulate the expression and regulation of some genes in related tissues by binding with some DNA or interacting with some proteins. Transcriptional regulatory factors are involved in organisms to determine which tissues and developmental stages of genes begin to transcribe [Berg JM, Shi Y. et al., Science, 1996, 271: 1081-1085] 0 Genes encoding such proteins If a mutation occurs, not only the gene itself cannot be expressed normally, but also many genes regulated by it cannot be normally transcribed and expressed. There are many types of transcriptional regulatory factors in the body, and the zinc finger protein and the bZIP protein superfamily are one of these proteins.
  • Zinc finger proteins are a class of proteins containing zinc atoms, and their action in vivo needs to be mediated by zinc atoms; while the role of bZIP proteins in the body needs to be mediated by leucine structure.
  • Zinc finger proteins can be divided into various types according to their structure different from zinc fingers, such as C2H2 type, CCCH type, CCHC type, C3HC4 type and so on. Among them, the C3HC4 zinc finger protein is also called ring finger protein. This type of protein is a new type of zinc finger protein. They differ from the general zinc finger binding proteins previously found in the number of metals, gaps, and sequence homology. Its protein structure contains multiple ring finger motifs.
  • the ring finger motif can be simply defined as Cys-X2-Cys-X (9-39) -Cys-X (1-3)-Hi s- X (2-3) -Cys-X2-Cys-X (4 -48)-Cys-X2-Cys, where X is any one amino acid (Freemont PS, Ann NY Acad Sci 1993, 684: 174-192). Studies have found that there are two main sequences that are similar to the ring finger protein motif. A domain called LIM contains eight conserved metal ligands with gaps similar to ring finger proteins, but with greater sequence conservation between individual metals (Schwabe JWR, lug A, Nat St ruct Biol, 1994, 1: 345-349). Another domain, called PHD or LAP, also contains eight conserved metal ligands and is similar to the LTM domain and ring finger motif (Aas l and R. et al., Trends Biochem Sci 1995, 20: 56-59).
  • the protein sequence of members of the C3HC4-type zinc finger protein family also contains a conserved "cros s-brace” structural motif, which is a linking system between proteins and zinc atoms.
  • cros s-brace a conserved "cros s-brace” structural motif, which is a linking system between proteins and zinc atoms.
  • Several conservative amino acid-based packages that form the hydrophobic core of the active protein configuration can be 01 00082 is common across all ring finger protein families. Therefore, the ring-finger motif forms a convenient scaffold system, and the structural changes of this system reflect the diversity of the functions of the family of protein molecules.
  • the two constituent domains of ring-finger proteins work together in vivo, and are responsible for participating in and regulating the transcription and expression of various related tissue genes, the aggregation of various protein macromolecules in the cell, and the interaction between regulatory proteins and proteins. A physiological process.
  • the ring finger protein domain acts as a potential homo-oligomerization domain with other target proteins to complete its regulation in vivo.
  • bZIP protein superfamily is another type of DNA-binding transcription factors found in eukaryotes.
  • the protein sequences of members of this protein family contain the conserved sequence fragments as follows: Consistent sequence fragments: [KR]-X ( 1, 3)-[RKSAQ] -NX (2)-[SAQ] (2) -X- [RKTAENQ] -XRX- [R]; Most members of the bZIP protein superfamily contain this conserved sequence fragment
  • the sequence fragment forms a conserved leucine zipper structure in the protein structure and mediates the interaction between the protein and DNA to form a protein configuration with regulatory activity and promotes the protein to complete its regulatory role in the body. This structural motif is expressed in various tissues and cells of the organism, and it participates in and regulates the transcription and expression of genes in various related tissues. Mutations in this sequence fragment are usually closely related to the occurrence of various immune disorders in the organism, tumors of related tissues and cancer.
  • the novel human transcriptional regulatory factor of the present invention contains both the above-mentioned conserved C3HC4 type zinc finger protein domain and the sequence fragment conserved by the bZIP protein superfamily, which is a new transcriptional regulatory factor in humans and regulates in vivo Transcription and expression of various related tissue genes.
  • the protein is involved in regulating the transcription and expression of various immune system and reproductive system-related genes in the body. Its abnormal expression is usually associated with some developmental disorders, reproductive system diseases, various immune disorders and tumors of related tissues. The occurrence of cancer is closely related. It can also be used to diagnose and treat various diseases mentioned above.
  • the human transcription regulator 80 protein plays an important role in important functions in the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more human transcription regulator 80 proteins involved in these processes. In particular, the amino acid sequence of this protein is identified. Isolation of the novel human transcriptional regulatory factor 80 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for the disease, so isolating its coding for DM is very important. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human transcriptional regulator 80.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human transcriptional regulator 80.
  • Another object of the present invention is to provide a method for producing human transcription regulator 80.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human transcriptional regulator 80.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human transcriptional regulator 80.
  • 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 61-2241 in SEQ ID NO: 1; and (b) a sequence having 1-3529 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said 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 the human transcriptional regulator 80 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to ⁇ The compound.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of human transcriptional regulator 80 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a biological sample The amount or biological activity of a polypeptide of the invention.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human transcriptional regulator 80.
  • 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 protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of 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 means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • Antagonist means a protein that, when combined with human transcriptional regulator 80, causes the protein to change from And molecules that regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind human transcriptional regulator 80.
  • Antagonist refers to a molecule that blocks or regulates the biological or immunological activity of human transcriptional regulator 80 when combined with human transcriptional regulator 80.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind human transcriptional regulator 80.
  • Regular refers to a change in the function of human transcription regulator 80, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune changes in human transcription regulator 80.
  • 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 transcription regulatory factor 80 using standard protein purification techniques.
  • Essentially pure Human Transcription Regulator 80 can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of human Transcription Regulator 80 polypeptide can be analyzed by amino acid sequence.
  • “Complementary” or “complementary” refers to the natural binding of a nucleotide by base-pairing under conditions of acceptable salt concentration and temperature.
  • the sequence "C-T-G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementation 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 completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to 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., Madi son Wis.). The MEGALIGN program can compare two or more sequences based on different methods such as the Clus ter method (Higg ins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method compares each pair by checking the distance between all pairs. Group sequences 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: Number of matching residues between sequence A and sequence X 100
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by a method known in the art such as Jotun Hein. (He in 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 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. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and?, which can specifically bind to the antigenic determinants of human transcription regulator 80.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, 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 part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated human transcription regulator 80 means that human transcription regulator 80 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human transcriptional regulator 80 using standard protein purification techniques. Non-reducing polymer A single main band can be produced on an acrylamide gel. The purity of the human transcriptional regulator 80 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a human transcriptional regulatory factor 80, which basically consists 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 invention can be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human transcriptional regulator 80.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human transcriptional regulator 80 of the invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or ( ⁇ ⁇ )
  • Such a type in which the mature polypeptide is fused with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • UV a type in which the additional amino acid sequence is fused into the mature polypeptide and formed by the polypeptide sequence ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found in a c library of human fetal brain tissue. It contains a polynucleotide sequence of 3529 bases in length and its open reading frame 61-2241 encodes 726 amino acids.
  • This polypeptide has the characteristic sequences of the C3HC4 type zinc finger protein domain and the bZIP protein superfamily. It can be deduced that the human transcriptional regulatory factor 80 has the structure and function represented by the C3HC4 type zinc finger protein domain and the bZIP protein superfamily.
  • the polynucleotide of the present invention may be in the DM form or the RM form.
  • DM forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DM can be a coding chain or a non-coding chain.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant” refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; The coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequences) of the mature polypeptide and non-coding sequences.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes 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.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (with at least two sequences between
  • 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 denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol 1, 42 ° C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity 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 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 transcriptional regulator 80.
  • 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 transcription regulator 80 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. Way to get (Qiagene). CDM libraries are also commonly used (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.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DM-RNA hybrids; (2) the presence or absence of marker gene functions; (3) measuring the level of human transcription regulator 80 transcripts; (4) Detection of gene-expressed protein products by immunological techniques or determination of biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect protein products expressed by the human transcriptional regulatory factor 80 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a human transcriptional regulatory factor 80 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding the human transcriptional regulatory factor 80 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, reverse transcription, as is well known in the art. Virus or other vector.
  • Vectors suitable for use in the present invention include, but are not limited to:
  • T7 promoter expression vector (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vector (Lee and Na thans, J Bi o 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 origins of replication, promoters, marker genes, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human transcription regulator 80 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, e.t. a. Mol. Molecular Cloning, a Labora tory Manua, Co., Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRM synthesis. Representative examples of these promoters are: the l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the origin of replication, the polyoma enhancer on the late side of the origin of replication, and the adenoviral 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 transcriptional regulatory factor 80 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or a recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transforming a host cell with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence may This is done using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human transcription regulator 80 (Scence, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • Figure 1 is a comparison diagram of the amino acid sequences of the human transcriptional regulatory factor 80 and C3HC4 type zinc finger protein domain and the bZIP protein superfamily functional domain of the present invention.
  • FIG. 1 Polyacrylamide gel electrophoresis (SDS-PAGE) Figure 1 is an isolated human transcription factor of 80 o 80 Da to the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDM sequence of one of the clones 0889e08 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the sequence of the human transcription regulatory factor 80 of the present invention and the protein sequence encoded by the same were used in a profiling scan program (Basic local Information search tool) in GCG [Alt schul, SF et al. J. Mol. Biol 1990; 215: 403-10], domain analysis was performed in databases such as Prote.
  • the human transcriptional regulatory factor 80 of the present invention is homologous to the domain C3HC4 type zinc finger protein domain and the bZIP protein superfamily, and the homology results are shown in FIG. 1.
  • Example 3 Cloning of a gene encoding human transcription regulator 80 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction.
  • PCR amplification was performed with the following primers:
  • Pr imerl 5'- GGGCGAAGAGTAGCGGTAGGTCGG -3 '(SEQ ID NO: 3)
  • Pr imer2 5'- AGTGCTTATGACATTCTTTATTCA -3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence starting at the Ibp at the 5 ′ end of SEQ ID NO: 1; Priraer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l KC1, 10 mmol / L Tris-CI, (pH8.5), 1.5 ⁇ l / L MgCl 2 , 200 ⁇ raol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -actin was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 3529bp shown in SEQ ID NO: 1.
  • Example 4 Northern 'blot analysis of human transcription regulator 80 gene expression:
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) and RM-transferred nitrocellulose membrane were placed in a solution at 42 ° C. C hybridization overnight, the solution contains 50% 'formamide-25raM KH 2 P0 4 (pH7.4) -5 ⁇ SSC-5 ⁇ Denhardt's solution and 200 ⁇ g / ml salmon sperm DM. After hybridization, filter was placed in 1 x SSC-0.1% SDS at 55. C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human transcriptional regulator 80
  • Primer 3 5'- CATGCTAGCATGGCTCAGACCGTGCGGAATGTT -3 '(Seq ID No: 5)
  • Primer 4 5'- CCCGAGCTCTCAACTTTTAGTTGCTTTTGATGG -3' (Seq ID No: 6)
  • the two ends of these two primers contain Nhel and Sacl digestion sites, respectively Points, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Nhel and Sacl restriction sites correspond to the selectivity on the expression vector plasmid pET 28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • the PCR reaction was performed using the pBS-0889e08 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are: pBS- ⁇ ⁇ ⁇ in a total volume of 50 ⁇ 1 Plasmid 10pg, 3
  • Nhel and Sacl were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into the colibacillus DH5 ⁇ by the calcium chloride method, and cultured overnight in LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), and then positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0889e08) 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-0889e08) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L, and continued Incubate for 5 hours.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation.
  • the purified human protein transcription factor 80 was purified.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following human transcription regulator 80-specific peptides: NH2-Met-Ala-Gln-Thr-Val-Arg-Asn-Yal-Thr-Leu-Ser-Leu-Thr- Leu-Pro- C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments 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, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt): 5 '-TGGCTCAGACCGTGCGGAATCTTACATTGTCGCACTACTG -3' (SEQ ID NO: 9)
  • probe 2 which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt): 5 '-TGGCTCAGACCGTGCGGAATCTTACATTGTCGCACTACTG -3' (SEQ ID NO: 9)
  • PBS phosphate buffered saline
  • 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 probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the sample film was placed in a plastic bag pre-hybridization solution was added 3- 10mg (10xDenhardt's; 6 xSSC, 0. lmg / ml CT DNA (calf thymus DNA). ). After sealing the bag, shake at 68 ° C for 2 hours.
  • Gene chip or gene microarray is a new technology currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, 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 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 .
  • 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 amplified product was adjusted to a concentration of about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m. The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method steps are widely reported in the literature. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from normal laryngeal and laryngeal carcinoma by one-step method, and the mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP 5 - ⁇ - propar gy 1- 2 '-deoxyur i dine 5'-tr iphate coupled to Cy3 f luorescent dye (purchased from Amersham Phamacia Biotech) was used to label the mRNA of normal laryngeal tissue.
  • Cy5dUTP (5-Amino-propargyl-2'-deoxyuridine 5'- tr iphate coupled to Cy5 f luorescent dye (purchased from Amersham Phamacia Biotech) was used to label laryngeal cancer tissue mRNA, and the probe was prepared after purification.
  • Cy5dUTP 5-Amino-propargyl-2'-deoxyuridine 5'- tr iphate coupled to Cy5 f luorescent dye (purchased from Amersham Phamacia Biotech) was used to label laryngeal cancer tissue mRNA, and the probe was prepared after purification.
  • the probes from the above two types of tissues were hybridized with the chip in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Then scan with a ScanArray 3000 scanner (purchased from General Scanning, USA). The scanned images are analyzed by I ma gene software (Biodi scovery, USA) to calculate the Cy3 / Cy5 ratio of each point, which is less than 0.5 points greater than 2 are considered tables Reach different genes.
  • polypeptides of the present invention 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.
  • Transcription regulators usually regulate the expression and regulation of some genes in related tissues by binding with some DNA or interacting with some proteins.
  • the zinc finger protein and the bZIP protein superfamily are a class of transcriptional regulatory factors.
  • the C3HC4 zinc finger protein binds to related proteins or nucleic acids through the C3HC4 ring finger domain and a conserved characteristic "cros s-brace" structural motif to regulate the transcription and expression of related genes.
  • C3HC4 zinc finger protein and immunoglobulin function has found that C3HC4 zinc finger protein and immunoglobulin function, interleukin-2 receptor, HIV-1 LTR promoter region gene expression, CDK kinase and cyclin H complex activity, sperm cell development, tyrosine kinase Activity, breast cancer, and multiple tumorigenesis are closely related.
  • the polypeptide of the invention contains a characteristic C3HC4 ring finger domain and a conserved characteristic "cros s-brace” structural motif, and also contains a conserved leucine zipper structure in the bZIP protein superfamily.
  • the conserved leucine zipper structure in the bZIP protein superfamily mediates protein-DNA interactions to form a protein configuration with regulatory activity and participates in and regulates the transcription and expression of genes in various related tissues. Mutations in this sequence are usually It is related to the occurrence of various immune disorders, tumors and cancers in related tissues.
  • the abnormal expression of the human transcriptional regulatory factor 80 of the present invention will produce various diseases, especially various tumors, immune system diseases, endocrine diseases, developmental disorders, and hematological malignancies. These diseases include, but are not limited to:
  • Tumors of various tissues breast cancer, endometrial cancer, thyroid tumors, uterine fibroids, neuroblastoma, ependymal tumors, colon cancer, leukemia, lymphoma, malignant histiocytosis, melanoma, sarcoma, bone marrow Tumor, teratoma, adrenal cancer, bladder cancer, bone cancer, bone marrow cancer, brain cancer, gallbladder cancer, liver cancer, lung cancer, thymic tumor 'immune diseases: rheumatoid arthritis, chronic active hepatitis, post-infection myocarditis, Systemic lupus erythematosus, scleroderma, myasthenia gravis, Guillain-Barre syndrome, autoimmune hemolytic anemia, primary B lymphocyte immunodeficiency disease, primary T lymphocyte immunodeficiency disease, primary Phagocytic immunodeficiency, primary complement system deficiency, acquired immunodeficiency syndrome, bron
  • Endocrine diseases diabetes insipidus, precocious puberty, giant disease and acromegaly, dwarfism, pituitary tumors, simple goiter, thyroiditis, hyperthyroidism, hypothyroidism, hyperparathyroidism, Hypoparathyroidism, hypercortisolism, primary hyperaldosteronism, adrenal insufficiency, adrenal tumors, diabetes, insulinoma, gastrinoma, premenstrual stress, menopausal syndrome, ovaries Hypoplasia, amenorrhea, male hypogonadism, precocious puberty
  • Abnormal expression of the human transcriptional regulatory factor 80 of the present invention will also cause certain hereditary diseases, blood diseases.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially various tumors, immune system diseases, endocrine diseases, developmental disorders, and bloody. Malignant diseases, certain genetic diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human transcriptional regulator 80.
  • Agonists enhance biological functions such as human transcriptional regulator 80 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human transcription regulator 80 can be cultured together with labeled human transcription regulator 80 in the presence of a drug.
  • 'Antagonists of human transcriptional regulator 80 include antibodies, compounds, receptor deletions and analogs that have been screened. Antagonists of human transcription regulator 80 can bind to human transcription regulator 80 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human transcriptional regulator 80 can be added to bioanalytical assays to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between human transcriptional regulator 80 and its receptor. 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 transcription regulator SO can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the human transcriptional regulatory factor 80 molecule 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.
  • antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human transcriptional regulatory factor 80 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 transcriptional regulator 80 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human transcriptional regulatory factor 80 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridization Tumor technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that combine human constant regions with non-human-derived variable regions can be produced using existing 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 transcription regulator 80.
  • Anti-Human Transcription Regulator 80 antibodies can be used in immunohistochemical techniques to detect human Transcription Regulator 80 in biopsy specimens.
  • Monoclonal antibodies that bind to human transcriptional regulator 80 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 transcription regulatory factor 80 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 toxin is bound to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human Transcription Regulator 80 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human transcriptional regulator 80.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human transcription regulator 80.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human transcription regulator 80.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human transcription regulator 80 detected in the test can be used to explain the importance of human transcription regulator 80 in various diseases and to diagnose diseases in which human transcription regulator 80 plays a role.
  • the 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 transcriptional regulator 80 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 transcription regulator 80.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human transcription regulator 80 to inhibit endogenous human transcription regulator 80 activity.
  • a variant human transcriptional regulatory factor 80 may be a shortened human transcriptional regulatory factor 80 that lacks a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human transcription regulator 80.
  • 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 transcriptional regulatory factor 80 into cells.
  • a recombinant viral vector carrying a polynucleotide encoding human transcriptional regulator 80 can be found in existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human transcription regulator 80 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RM and DNA
  • ribozymes that inhibit human transcription regulator 80 mRNA 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 A for endonucleation.
  • Antisense RNA, DNA and ribozymes can be obtained by any RNA or DNA synthesis technology, such as solid-phase phosphoramidite synthesis of oligonucleotides, and are 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.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human transcription regulator 80 can be used for the diagnosis of diseases related to human transcription regulator 80.
  • the polynucleotide encoding human transcription regulator 80 can be used to detect the expression of human transcription regulator 80 or the abnormal expression of human transcription regulator 80 in a disease state.
  • a DNA sequence encoding human transcription regulator 80 can be used to hybridize biopsy specimens to determine the expression of human transcription regulator 80.
  • 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 used as probes to be fixed on a micro array or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human Transcription Regulator 80 specific primers can also be used to detect the transcription products of human Transcription Regulator 80 by RNA-polymerase chain reaction (RT-PCR) in vitro amplification.
  • RT-PCR RNA-polymerase chain reaction
  • Human Transcription Regulator 80 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human Transcription Regulator 80 DNA sequences. Mutations can be detected using existing techniques such as Southern blotting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • 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, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the 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 staining Structural changes in the body, such as deletions or translocations that are visible from the chromosomal level or detectable with cDM sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the CDM that is accurately mapped to a disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human transcriptional regulator 80 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human transcription regulator 80 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, un facteur régulateur 80 de la transcription humaine, 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 le facteur régulateur 80 de la transcription humaine.
PCT/CN2001/000082 2000-01-28 2001-01-21 Nouveau polypeptide, facteur regulateur 80 de la transcription humaine, et polynucleotide codant pour ce polypeptide Ceased WO2001055381A1 (fr)

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CN 00111582 CN1307044A (zh) 2000-01-28 2000-01-28 一种新的多肽——人转录调节因子80和编码这种多肽的多核苷酸
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Publication number Priority date Publication date Assignee Title
WO2014191575A1 (fr) * 2013-05-31 2014-12-04 Centre National De La Recherche Scientique (Cnrs) Protéine impliquée dans la réplication de l'adn, et modulation de son activité

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EUR. J. IMMUNOL., vol. 20, no. 6, June 1990 (1990-06-01), pages 1337 - 1343 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014191575A1 (fr) * 2013-05-31 2014-12-04 Centre National De La Recherche Scientique (Cnrs) Protéine impliquée dans la réplication de l'adn, et modulation de son activité

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