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WO2002040522A1 - NOUVEAU POLYPEPTIDE, PROTEINE HUMAINE D'ACTIVATION 14.08 DU FACTEUR β DE REGULATION DE LA CROISSANCE ET DE TRANSCRIPTION, ET POLYNUCLEOTIDE CODANT CE POLYPEPTIDE - Google Patents

NOUVEAU POLYPEPTIDE, PROTEINE HUMAINE D'ACTIVATION 14.08 DU FACTEUR β DE REGULATION DE LA CROISSANCE ET DE TRANSCRIPTION, ET POLYNUCLEOTIDE CODANT CE POLYPEPTIDE Download PDF

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Publication number
WO2002040522A1
WO2002040522A1 PCT/CN2001/001096 CN0101096W WO0240522A1 WO 2002040522 A1 WO2002040522 A1 WO 2002040522A1 CN 0101096 W CN0101096 W CN 0101096W WO 0240522 A1 WO0240522 A1 WO 0240522A1
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Prior art keywords
polypeptide
polynucleotide
growth regulator
human
protein
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PCT/CN2001/001096
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU2002210330A priority Critical patent/AU2002210330A1/en
Publication of WO2002040522A1 publication Critical patent/WO2002040522A1/fr

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

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide-to-human transcription growth regulator P-activator protein 14. 08, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • the transcription and expression of genes are jointly regulated by a variety of proteins, which constitute a large family of proteins in the body, namely the transcription factor protein family.
  • Members of this protein family bind to specific DNA sequences to regulate gene transcription and expression.
  • These DM-binding domains have roughly four fancy structures; alpha helix-turn-alpha helix, Cys-His zinc fingers, Cys-Cys zinc fingers, and Leu zippers. These crust formations are important for maintaining the structure of the entire domain and the activity of proteins in vivo.
  • Transcriptional growth regulatory factor P is also an important transcriptional regulatory factor involved in regulating the transcription and expression of various genes in the body. The activity of this protein factor in the body is regulated by a variety of related proteins, such as related activating proteins and upstream regulatory proteins.
  • bladder mucosa PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 1024NT, scar-like fc growth factor stimulation, 1013HT, scar Fc not stimulated with growth factors, 1013HC, bladder
  • bladder cancer bladder cancer, liver cancer, liver cancer cell lines, placenta, spleen, prostate cancer, jejunum adenocarcinoma, and cardia cancer
  • the expression profiles of the proteins are very similar, so the functions of the two may be similar.
  • the invention is named human transcription growth regulator beta-activating protein 14. 08.
  • the human transcription growth regulator P-activating protein 14. 08 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, so identification in the art has been required More human transcription growth regulator P activating protein 14. 08 proteins involved in these processes, especially the amino acid sequence of this protein was identified.
  • New human transcriptional growth regulator ⁇ -activating protein 14. The isolation of the 08-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 diseases, so isolating its coding DNA is important.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human transcriptional growth regulator ⁇ -activating protein 14. 08.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human transcriptional growth regulator ⁇ -activating protein 14. 08.
  • Another object of the present invention is to provide a method for producing human transcriptional growth regulator ⁇ -activator protein 14. 08.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human transcriptional growth regulator P-activating protein 14.08.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, a human transcriptional growth regulator P-activating protein 14.08.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormality of human transcriptional growth regulator P-activating protein 14.08. Summary of invention
  • 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 106 to 492 in SEQ ID NO: 1; and (b) a sequence having 1-2057 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 the polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human transcriptional growth regulator ⁇ -activating protein 14. 08 protein, which comprises using the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for in vitro detection of a disease or disease susceptibility associated with abnormal expression of human transcriptional growth regulator P-activating protein 14.08 protein, which comprises detecting the presence of the polypeptide or its encoding polynucleotide sequence in a biological sample. Mutates, or detects the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the present invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the preparation of a polypeptide and / or polynucleotide of the present invention for the treatment of cancer, developmental disease or immune disease or other drugs caused by abnormal expression of human transcriptional growth regulator P activating protein 14. 08 use.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human transcription growth regulator ⁇ -activator I 4 ⁇ 08 and human transcription growth regulator ⁇ -activator of the present invention.
  • the upper graph is a graph of the expression profile of human transcription growth regulator beta-activating protein 14. 08, and the lower graph is the graph of the expression profile of human transcription growth regulator beta-activating protein.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human transcriptional growth regulator P-activator protein 14. 08. 14kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. Summary of the Invention
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or RM, 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 being replaced 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 transcriptional growth regulator beta-activating protein 14.08, 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 human transcriptional growth regulator beta-activating protein 14.08.
  • "Antagonist” or “inhibitor” refers to a biological activity or immunity that can block or regulate human transcriptional growth regulator P-activating protein 14.08 when combined with human transcriptional growth regulator-beta activating protein 14.08.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human transcriptional growth regulator P-activating protein 14.08.
  • Regular refers to any change in the function of human transcription growth regulator P-activating protein 14. 08, including an increase or decrease in protein activity, changes in binding characteristics, and any other organism of human transcription growth regulator P-activating protein 14. 08 Changes in nature, function, or immunity.
  • substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art 3 ⁇ 4 Purify human transcriptional growth regulator ⁇ -activating protein 14. 08 using standard protein purification techniques. Essentially pure human transcriptional growth regulator P-activating protein 14. 08 produces a single main band on a non-reducing polyacrylamide gel. Human transcriptional growth regulator ⁇ -activator protein 14. 08 Purity of peptides 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 can 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. Inhibition of such 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 conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are the same or similar in a comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higg ins, D. G. and P. M. Sharp (1988)
  • the Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (Hein L, (1990) Methods in enzymology 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 substitution 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 HFP or a chemical modification of its nucleic acid. Such a chemical modification may be a substitution of a hydrogen atom with a fluorenyl 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? It can specifically bind to the epitope of human transcription growth regulator ⁇ -activating protein 14. 08.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living 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 certain vector, or such a polynucleotide or polypeptide may be part of a certain 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 transcriptional growth regulator beta-activating protein 14. 08 means human transcriptional growth regulator beta-activating protein 14. 08 is substantially free of other proteins, lipids, carbohydrates, or others that are naturally associated with it. substance. Those skilled in the art can use standard protein purification techniques to purify human transcription growth regulator ⁇ -activating protein 14. 08. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human transcriptional growth regulator ⁇ -activating protein 14. 08 polypeptide can be analyzed by amino acid sequence. The present invention provides a new polypeptide, human transcription growth regulator ⁇ -activating protein 14. 08, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of the human transcriptional growth regulator P-activator protein 14.08.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human transcriptional growth regulator P-activating protein 14.08 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide ( Such as leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences).
  • 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 a 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 2057 bases in length and its open reading frame 106-492 encodes 128 amino acids.
  • this polypeptide has a similar expression profile with human transcription growth regulator P-activating protein, and it can be inferred that the human transcription growth regulator ⁇ -activating protein 14. 08 has similar human transcription growth regulator ⁇ -activating protein. Functions.
  • the polynucleotide of the present invention may be in the form of DNA or RM.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • 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 (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) with denaturing agents, such as 50% (v / v) formamide, 0.1% calf serum / 0.1 lcol, 42 ° C, etc .; or (3 ) Hybridization occurs only when the identity between the two sequences is at least 95% or more, and more preferably 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide and the mature polypeptide shown in SEQ ID NO: 2 Have the same biological 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 transcriptional growth regulator ⁇ -activator protein 14. 08.
  • 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 transcriptional growth regulator ⁇ -activating egg 14. 08 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the DM of the genome; 2) chemically synthesizing the DM sequence to obtain the double-stranded DM of the polypeptide.
  • genomic DNA isolation is the least commonly used.
  • the direct chemical synthesis of the DM sequence is Often chosen method.
  • 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.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold. Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be screened from these cDM libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determination of the human transcriptional growth regulator ⁇ -activator protein 14. 08 transcript (4) Detecting the protein product of gene expression by immunological techniques or measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is 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 transcription growth regulator ⁇ -activating protein 14. 08 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) .
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) .
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RM 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 vector using the vector of the present invention or directly used Human transcriptional growth regulator beta-activating protein 14. 08 coding sequence, genetically engineered host cells, and a method for producing the polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding a human transcriptional growth regulator ⁇ -activating protein 14. 08 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, 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 expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct an expression vector containing a DM sequence encoding human transcriptional growth regulator beta-activating protein 14.08 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant technology (Sambroook, et a l. Molecular Cloning, a Laboratory Manual, Cold Spr ing Harbor Laboratory. New York, 1989) the DNA sequence is operably linked 0 To the appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site 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. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a human transcription growth regulator P activating protein 14. 08 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute the polynucleotide or the recombinant vector.
  • Genetically engineered host cells 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 Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with (1 2 method used in the step are well known in the art. Alternatively, it is a MgCl 2. If 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 Wait.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human transcriptional growth regulator ⁇ -activating protein 14. 08 (Scence, 1984; 224: 1431). Generally speaking, 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 outside the cell.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • Transcriptional growth regulator P is an important transcriptional regulator, which is involved in regulating the transcription and expression of various genes in the body.
  • the activity of this protein factor in the body is regulated by a variety of related proteins. Such as related activating proteins.
  • the human transcriptional growth regulator P activating protein binds to the transcriptional growth regulator P in vivo and activates its activity in vivo to regulate the transcription and expression of various related genes. Its abnormal expression can cause the body to regulate the transcription and expression of various genes, and then cause related diseases.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human transcriptional growth regulator P-activating protein, and both have similar biological functions.
  • the polypeptide of the present invention is combined with transcriptional growth regulatory factor ⁇ in vivo and activates its action activity in vivo to regulate the transcription and expression of various related genes. Abnormal expression can cause the body to regulate the transcription and expression of various genes, which can lead to the development of embryonic malformations, tumors, and disorders of protein metabolism. These diseases include, but are not limited to:
  • Cleft lip (most common, with alveolar cleft and cleft palate), cleft lip, facial oblique cleft, cervical pouch, cervical fistula, etc.
  • Horizontal absence congenital short limbs: no arms, no forearms, no hands, no fingers, no legs, no toes, etc .; longitudinal absences: upper limb radius / ulnar insufficiency, lower limb tibial / fibula insufficiency, etc .;
  • Limb differentiation disorder lack of a certain muscle or muscle group, joint dysplasia, bone deformity, bone fusion, multi-finger (toe) deformity, and (toe) malformation, horseshoe varus, etc .;
  • Thyroglossal duct cysts atresia or stenosis of the digestive tract, ileal diverticulum, umbilical diaphragm, congenital umbilical hernia, congenital agangliomegalo colon, impotence of anus, abnormal bowel transition, bile duct atresia, circular pancreas, etc
  • Neural Tube Defects (Anecephalic Malformations, Spina bifida, Spinal Meningocele Outflow, hydrocephalus meningocele), hydrocephalus in / outside the brain, etc .;
  • Papilloma squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (carcinoma) [breast, thyroid], mucinous / serous cystadenomas (carcinoma) [ovary], basal cell carcinoma [head and face Skin], (malignant) polytype adenoma [extending gland], papilloma, transitional epithelial cancer [bladder, renal pelvis], etc .;
  • Malignant lymphoma [Neck, mediastinum, mesenteric and retroperitoneal lymph nodes], various leukemias [lymphoid hematopoietic tissue], multiple myeloma [push / thoracic / rib / skull and long bone], etc .;
  • Nerve fiber [systemic cutaneous nerve / deep nerve and internal organs], (malignant) schwannoma [nervous of head, neck, limbs, etc.], (malignant) glioblastoma [brain], myeloblastoma Cerebellum], (malignant) meningiomas [meninges], ganglioblastoma / neuroblastoma [mediastinum and retroperitoneum / adrenal medulla], etc .;
  • malignant melanoma skin, mucous membrane
  • (malignant) hydatidiform mole chorionic epithelial cancer [uterine]
  • (malignant) supporter cells stromal cell tumor
  • (malignant) granulosa cell tumor ovarian, testicular] fine Blastoma [testis], asexual cell tumor '[ovary], embryonal cancer [testis, ovary], (malignant) teratoma [ovary, testis, mediastinum and palate tail], etc .
  • malignant melanoma skin, mucous membrane
  • hydatidiform mole chorionic epithelial cancer [uterine]
  • (malignant) supporter cells stromal cell tumor
  • (malignant) granulosa cell tumor ovarian, testicular] fine Blastoma [testis]
  • asexual cell tumor '[ovary] embryonal cancer [testis, ovary]
  • Protein peptide hormone dysfunction can cause the following diseases:
  • Insulin and glucagon diabetes, hypoglycemia, etc .;
  • hypothalamus and pituitary hormones Giant disease, dwarfism, acromegaly, Cortisol syndrome (Cushing's syndrome), primary hyperaldosteronism, secondary chronic adrenal insufficiency, hyperthyroidism Hypothyroidism (stingle disease, juvenile hypothyroidism, adult hypothyroidism), male / female infertility, menstrual disorders (functional uterine bleeding, amenorrhea, polycystic ovary syndrome, premenstrual tension syndrome, Menopause syndrome), sexual development disorder, diabetes insipidus, inappropriate antidiuretic hormone secretion syndrome, abnormal lactation, etc .;
  • parathyroid hormone hyperparathyroidism, hypoparathyroidism, etc .
  • Gastrointestinal hormones peptic ulcer, chronic indigestion, chronic gastritis, etc .;
  • Arrhythmia shock, insanity, epilepsy, chorea, hepatic encephalopathy (norepinephrine, Y-aminobutyric acid, serotonin, glutamine), motion sickness, type I allergic disease (Net Measles, hay fever, allergic rhinitis, skin allergies), peptic ulcer (histamine), hypercholesterolemia (taurine), tumors (polyamines), etc .;
  • the polypeptide of the present invention and the antagonist, agonist and inhibitor of the polypeptide can be directly used in the treatment of various diseases, such as embryonic developmental malformations, tumors, and disorders of protein metabolism.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human transcriptional growth regulator P-activator protein 14. 08.
  • Agonists increase human transcriptional growth regulator ⁇ -activating protein 14. 08 stimulates biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human transcriptional growth regulator P activating protein 14. 08 can be cultured with a labeled human transcriptional growth regulator P activating protein 14. 08 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human transcription growth regulator ⁇ -activating protein 14. 08 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human transcription growth regulator P activating protein 14. 08 can bind to human transcription growth regulator P activating protein 14. 08 and eliminate its function, or inhibit the polypeptide Production, or binding to the active site of the polypeptide prevents the polypeptide from performing biological functions.
  • human transcription growth regulator ⁇ -activating protein 14. 08 can be added to the bioanalytical assay, and by measuring the compounds against human transcription growth regulator ⁇ -activating protein 14. 08 and their receptors, Effect 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.
  • 3 activator protein 14. 08 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 growth regulator P activator protein 14. 08 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.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human transcriptional growth regulator ⁇ -activating protein 14. 08 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 obtained by direct injection of human transcriptional growth regulator ⁇ -activating protein 14. 08 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not Limited to Freund's adjuvant and the like.
  • Techniques for preparing monoclonal antibodies to human transcription growth regulator ⁇ -activating protein 14. 08 include, but are not limited to, hybridoma technology (Kohler 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 to non-human 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 transcriptional growth regulator ⁇ -activator protein 14. 08.
  • Antibodies against human transcriptional growth regulator ⁇ -activator protein 14. 08 can be used in immunohistochemical techniques to detect human transcriptional growth regulator P-activator protein 14. 08 in biopsy specimens.
  • Monoclonal antibodies that bind to human transcriptional growth regulator P-activating protein 14. 08 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.
  • 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 transcriptional growth regulator P activating protein 14 08 positive cells.
  • the antibodies of the present invention can be used to treat or prevent human transcriptional growth regulator P activating protein 14. 08 Related diseases. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human transcriptional growth regulator P-activating protein 14.08.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human transcriptional growth regulator P-activator protein 14.08. These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human transcription growth regulator P activator protein 14. 08 detected in the test can be used to explain the importance of human transcription growth regulator P activator protein 14. 08 in various diseases and to diagnose human transcription growth regulator Diseases where beta activin 14. 08 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry.
  • Polynucleotides encoding human transcriptional growth regulator P-activating protein 14. 08 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 transcriptional growth regulator ⁇ -activator protein 14. 08.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human transcription growth regulator ⁇ -activator protein 14. 08 to inhibit endogenous human transcription growth regulator ⁇ -activator protein 14. 08 activity.
  • a variant human transcriptional growth regulator ⁇ -activating protein 14. 08 may be a shortened human transcriptional growth regulator
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human transcriptional growth regulator ⁇ -activating protein 14.08.
  • 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 growth regulator ⁇ -activating protein 14. 08 into cells.
  • Methods for constructing recombinant viral vectors carrying polynucleotides encoding human transcriptional growth regulator beta-activating protein 14.08 can be found in existing literature (Sambrook, et al.).
  • the recombinant polynucleotide encoding human transcription growth regulator ⁇ -activator protein 14. 08 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 MA and DNA
  • ribozymes that inhibit human transcription growth regulator P activating protein 14. 08 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose a specific RNA, and its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RNA and DM and ribozymes can be obtained by any existing RNA or DNA synthesis technology. For example, the technology for the synthesis of oligonucleotides by solid-phase phosphate amide chemical synthesis has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated into the vector's RNA Downstream of the polymerase promoter. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human transcription growth regulator ⁇ -activator protein 14. 08 can be used for the diagnosis of diseases related to human transcription growth regulator ⁇ -activator protein 14. 08.
  • a polynucleotide encoding human transcription growth regulator ⁇ -activator protein 14. 08 can be used to detect the expression of human transcription growth regulator ⁇ -activator protein 14. 08 or human transcription growth regulator ⁇ -activator protein 14. 08 in a disease state. Abnormal expression.
  • the DNA sequence encoding human transcription growth regulator ⁇ -activator protein 14. 08 can be used to hybridize biopsy specimens to determine the expression of human transcription growth regulator ⁇ -activator protein 14. 08.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization.
  • polynucleotides of the present invention can be used as probes 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 tissues.
  • the transcription product of human transcription growth regulator ⁇ -activator protein 14. 08 can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using human transcription growth regulator ⁇ -activator protein 14. 08-specific primers.
  • Detection of mutations in the human transcriptional growth regulator ⁇ -activator protein 14. 08 gene can also be used to diagnose human transcriptional growth regulator ⁇ -activator protein 14. 08-related diseases.
  • Human transcriptional growth regulator beta-activating protein 14. 08 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human transcriptional growth regulator beta-activating protein 14. 08 DNA sequences. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern blotting and Wes tern blotting can be used to indirectly determine the presence or absence of mutations in a gene.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position of a human chromosome and can hybridize with it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeat polymorphisms) are available for labeling chromosomal positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on the cDNA, and the sequence can be mapped on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells containing human genes corresponding to the primers will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize MA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments or Genomic cloning to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cD libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all of the affected individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • 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 transcriptional growth regulator beta-activating protein 14. 08 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human transcriptional growth regulator beta-activating protein 14.08 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. Examples
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDM sequence was compared with an existing public DNA sequence database (Genebank :), and the cDNA sequence of one of the clones 1100a05 was found to be new DNA.
  • the inserted cDM fragments contained in this clone were assayed in both directions by synthesizing a series of primers.
  • CDNA was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification using Q i agene's kit, the following primers were used for PCR amplification:
  • Primerl 5,-AAGGAAAGATTATTCAGTTGGGGC -3, (SEQ ID NO: 3)
  • Pr imer2 5'- ACTTGAATGCTGTTACTATTTATT -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, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification reaction conditions containing 5 0mmol / L KC1 in a reaction volume of 50 ⁇ 1, lOmmol / L Tris-HCl pH8 5, 1. 5mmol / L MgCl 2, 200 ⁇ 1 / ⁇ dNTP, lOpmol primer, 1U of Taq DM polymerization. Enzyme (Clontech). Cycles were performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions: 94. C 30sec; 55. C 30sec; 72 ° C 2min. During RT-PCR, ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen) using a TA cloning kit.
  • DNA sequence analysis results indicate the DNA sequence of the PCR product It is exactly the same as 1-2057bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human transcription growth regulator ⁇ -activator protein 14.08 gene expression
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159].
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25raM sodium citrate, 0.2M sodium acetate (pH 4. ()), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) After mixing, centrifuge. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • Electrophoresis was performed on a 1.2% agarose gel containing 2 g of RNA on 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then Transfer to a nitrocellulose membrane. A 32 P dATP was used to prepare a 32 P-labeled DNA probe by a random primer method. The DM probe used was activated by the PCR-amplified human transcriptional growth regulator ⁇ shown in FIG. 1. Protein 14.08 coding region sequence (106bp to 492bp).
  • 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred in a solution at 42 ° C overnight.
  • the solution Contains 50% formamide-25mM KH 2 P0 4 (pH7. 4 ) -5 xSSC- 5 x Denhardt's solution and 200 ⁇ ⁇ ⁇ 1 salmon sperm DNA.
  • the filter is washed in 1 x SSC-0.1% SDS at 55 ° C. 30min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human transcription growth regulator ⁇ -activator protein 14.08
  • Primer3 5 '-CATGCTAGCATGAGAGAAGAAAAG AGAGAAGAA- 3' (Seq ID No: 5)
  • Primer4 5'-CATGGATCCCTAGCATGTATAAATCAAACATAA-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Nhel and BainHI digestion sites, respectively, followed by the coding sequences of the 5 'and 3, ends of the target gene, respectively.
  • the Nhel and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3).
  • the PCR reaction was performed using the pBS-1100a05 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-1100a05 plasmid, primer Primer-3 and Primer-4 points in a total volume of 50 ⁇ 1; 1 J was lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Nhel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into colibacillus DH5a by the calcium chloride method.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was subjected to centrifugation, and chromatography was performed using His s. Bind Quick Cartr idge (product of Novagen) which can bind 6 histidine (6His-Tag). 08.
  • the purified target human transcriptional growth regulator P-activator protein 14.08 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 14 kDa ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2.
  • Example 5 Production of anti-human transcriptional growth regulator P-activating protein 14. 08 antibody
  • NH2-Met-Arg-Glu-Glu-Lys-Arg-Glu-Glu-Arg-Arg-Arg-Glu-I le-Glu-C00H SEQ ID NO: 7
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin for methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the above-mentioned jk cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • the titer of antibody in rabbit serum was determined by ELISA using a titer plate coated with 15 ⁇ ⁇ / ⁇ 1 bovine serum albumin polypeptide complex.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibody could specifically bind to human transcriptional growth regulator P-activating protein 14. 08.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Nor thern blotting method and copying method, etc., are all used to fix the polynucleotide sample to be tested on the filter membrane and then hybridize using basically the same steps.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic 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 utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above 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):
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • the sample film was placed in a plastic bag pre-hybridization solution was added 3-10m g (lOxDenhardt's; 6xSSC, 0. Img / 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 .
  • the specific method steps have been reported in the literature.
  • cDM total of four full-length nucleotide sequence of more than 000 as a target DNA which comprises a 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 and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slide to prepare a chip.
  • Cartesian 7500 spotter purchased from Cartesian Company, USA. The distance between them is 280 ⁇ m.
  • the spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slide to prepare a chip.
  • the specific method steps are in the text There have been various reports in the offering.
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified with Oligotex mRNA Midi Ki t (purchased from QiaGen).
  • Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Amino-propargyl-2, -deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy3 f luorescent dye, purchased from Amersham P
  • 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. Scanning was then performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are fetal brain, bladder mucosa, PMA + EcWO 4 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 1024NT, scar Stimulation of fc growth factor, 1013HT, scar growth without fc stimulation, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma Cardiac cancer. Based on these 18 Cy3 / Cy5 ratios, a histogram is drawn (Figure 1). It can be seen from the figure that the expression profile of human transcription growth regulator P-activator protein 14. 08 and human transcription growth regulator P-activator protein according to the present invention are very similar.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine d'activation 14.08 du facteur β de régulation de la croissance et de transcription, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment de malformations apparaissant durant le développement de l'embryon, de tumeurs et de maladies liées aux troubles du métabolisme des protéines. 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 d'activation 14.08 du facteur β de régulation de la croissance et de transcription.
PCT/CN2001/001096 2000-06-30 2001-06-29 NOUVEAU POLYPEPTIDE, PROTEINE HUMAINE D'ACTIVATION 14.08 DU FACTEUR β DE REGULATION DE LA CROISSANCE ET DE TRANSCRIPTION, ET POLYNUCLEOTIDE CODANT CE POLYPEPTIDE WO2002040522A1 (fr)

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AU2002210330A AU2002210330A1 (en) 2000-06-30 2001-06-29 A novel polypeptide-homo transcription growth regulatory factor beta activation protein 14.08 and polynucleotide encoding said polypeptide

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CN00116905.X 2000-06-30
CN 00116905 CN1331199A (zh) 2000-06-30 2000-06-30 一种新的多肽——人转录生长调控因子β激活蛋白14.08和编码这种多肽的多核苷酸

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WO2002040522A1 true WO2002040522A1 (fr) 2002-05-23

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PCT/CN2001/001096 WO2002040522A1 (fr) 2000-06-30 2001-06-29 NOUVEAU POLYPEPTIDE, PROTEINE HUMAINE D'ACTIVATION 14.08 DU FACTEUR β DE REGULATION DE LA CROISSANCE ET DE TRANSCRIPTION, ET POLYNUCLEOTIDE CODANT CE POLYPEPTIDE

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WO (1) WO2002040522A1 (fr)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GUO YUANCHUN ET AL.: "Structure and function of transcription activation protein", BULLETIN OF BIOLOGY, vol. 30, no. 6, 1996, pages 13 - 15 *
WU JIANSHENG ET AL.: "Functional domains of the regulatory factor PHO81 of saccharomyces cerevisiae", ACTA BIOCHIMICA ET BIOPHYSICA SINCA, vol. 28, no. 5, September 1996 (1996-09-01), pages 507 - 515 *

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