WO2001029080A1 - Nouveau polypeptide, une proteine humaine hmg-13, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, une proteine humaine hmg-13, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001029080A1
WO2001029080A1 PCT/CN2000/000333 CN0000333W WO0129080A1 WO 2001029080 A1 WO2001029080 A1 WO 2001029080A1 CN 0000333 W CN0000333 W CN 0000333W WO 0129080 A1 WO0129080 A1 WO 0129080A1
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Prior art keywords
polypeptide
polynucleotide
high mobility
human high
component protein
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PCT/CN2000/000333
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Bio Road Gene Development Ltd.
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Priority to AU78993/00A priority Critical patent/AU7899300A/en
Publication of WO2001029080A1 publication Critical patent/WO2001029080A1/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
    • 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, human high mobility component protein 13, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background
  • HMG High Mobility Group
  • HMG is defined as a chromatin protein that:
  • the molecular weight is less than 30,000.
  • HMG can be divided into three different families: HMG-1 / 2 family, HMG-14 / 17 family and HMG-1 family. Among them, the HMG-1/2 family has the largest number.
  • the novel protein HMG-H protein of the present invention belongs to the HMG-1 / 2 family.
  • HMG-1 / 2 mRNAs with molecular weights of about 2.4kb, 1.4kb, and lkb, which may be different in cells of different origin, of which 1.4kb The highest mRNA content.
  • the biggest feature of HMG-1 / 2 mRNA is that the 3 'untranslated region is highly conserved in evolution, while its 5' untranslated region is not highly conserved.
  • the GC content is the same as the AT content, while in the untranslated region, A + T accounts for 66 ° /. .
  • the HMG-1 / 2 protein contains two domains: domain A (residues 1-79) and domain B (residues 90-163). These two domains are highly conservative in evolution, have a globular structure, and have a net charge of +20 because they are rich in charged amino acids.
  • the third part of the HMG-1 / 2 protein starts at the 164th amino acid and has a charge imbalance structure. The first 20 amino acids have a net charge of +8, and the last 29 amino acids form a negatively charged acid end.
  • HMG-1 / 2 can bind to double-stranded and single-stranded DNA, but more easily to single-stranded DNA. HMG-1 / 2 easily binds to negative supercoiled DNA, preventing the formation of specific secondary structures. The negatively charged C-terminal region of HMG1 / 2 appears to make DNA unwinding easier.
  • HMG1 / 2 protein is present in both the nucleus and the cytoplasm.
  • the inverse correlation between the level of HMG1 / 2 and the level of histone HI indicates that HMG1 / 2 Relationship between the distribution and cell proliferation and differentiation.
  • HMG protein is required for the transcription of chromatin. If the HMG protein is inhibited by antibodies, the transcription loop on the lamp brush chromosome will be retracted. However, further research shows that HMG protein is not a direct activator of transcription, but may keep the transcription part of chromatin in an activated conformation phase and easily bind to transcription activators [Bustin, Michael, Biochimica et Biophys ica Acta, 1049 ( 1990) 231-243] 0
  • the polypeptide of the present invention is inferred and identified as a high mobility component protein H (HMG-H). This result was obtained by amino acid homology comparison.
  • HMG-H high mobility component protein H
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human high mobility component protein 13.
  • Another object of the present invention is to provide a method for producing human high mobility component protein 13.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, a human high mobility component protein 13.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, human high mobility component protein 13.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human high mobility component protein 13. Summary of invention
  • a novel isolated human high mobility component protein 13 is provided.
  • the peptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof, or the like.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • Human high mobility component protein 1 3 is characterized by a kinase domain consisting of approximately 122aa.
  • a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 70 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human high mobility component protein 1 3; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 858-1226 in SEQ ID NO: 1; and (b) a sequence having 1-2446 in SEQ ID NO: 1 Sequence of bits.
  • Fig. 1 is a comparison diagram of amino acid sequence homology between the human high mobility component protein 13 and human HMG-2a of the present invention.
  • the upper sequence is human high mobility component protein 1 3 and the lower sequence is human HMG-2a.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human high mobility component protein 1 3. 13.5 kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human high mobility component protein 1 3 means that human high mobility component protein 1 3 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated.
  • Those skilled in the art can purify human high mobility component protein 13 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human high mobility component protein 13 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human high mobility component protein 13, 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 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 human high mobility component protein 1 3.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human high mobility component protein 1 3 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: U) a type in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted
  • the amino acid may or may not be encoded by the genetic code; or ( ⁇ ) such that a group on one or more amino acid residues is substituted by another group to include a substituent; or ( ⁇ ⁇ ) like this
  • 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
  • IV a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • an additional amino acid sequence is fused into a mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 2446 bases in length and its open reading frame (858-1226) encodes 122 amino acids.
  • this polypeptide has 64% homology with human HMG-2a, and the polypeptide has two conservative bases of the HMG-2a gene family, which can be inferred from this new human high mobility
  • the component proteins have similar structures and functions of the HMG-2a gene family.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDM, genomic DNA, or synthetic D.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only between the two sequences Crosses occur at least 95% or more, and more preferably 97% or more.
  • 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.
  • the "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 Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human high mobility component protein 13.
  • 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 high mobility component protein 13 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. Isolate cDNA of interest The standard method is to isolate raRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene). 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 selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DM-DNA or DNA-RM hybridization; (2) the presence or loss of marker gene function; (3) determining the level of human high mobility component protein 13 transcripts; (4) Detecting the protein product of gene expression by immunological technology or measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • 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 the protein product expressed by the human high mobility component protein 13 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 DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured 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 cD sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the human high mobility component protein 13 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding human high mobility component protein 13 may be inserted into a vector, In order to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct an expression vector containing a DNA sequence encoding human high mobility component protein 1 3 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, etal. Molecluar Cloning, a Labora tory Manua l, Col Spring Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers from 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 human high mobility component protein 1 3 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Representative examples are: Large Enterobacter, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf 9; animal cells such as CH0, COS or Bowes s melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be harvested after exponential growth phase, with (& (Treatment 1 2, procedure used are well known in the art. Alternatively, it is used MgC l 2. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following D transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipid Plastid packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human high mobility component protein 1 3 (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. If necessary, the recombinant protein can be isolated and purified by various separation methods using its 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
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat uterine leiomyoma, neuroblastoma, red leukemia, and mouse teratoma.
  • HMG is related to the growth and development of mammals. HMG may play an important role in uterine leiomyomas and may provide another treatment for this disease.
  • HMG protein can be used to detect these diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human high mobility component protein 13.
  • Agonists enhance human high mobility component protein 13 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing human high mobility component protein 13 can be cultured together with labeled human high mobility component protein 13 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human high mobility component protein 13 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human high mobility component protein 13 can bind to human high mobility component protein 13 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 function biological functions.
  • human high mobility component protein 13 When screening compounds as antagonists, human high mobility component protein 13 can be added to the bioanalytical assay, and the compound can be determined by measuring the effect of the compound on the interaction between human high mobility component protein 13 and its receptor Whether it is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to human high mobility component protein 13 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 13 molecules of human high mobility component protein should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human high mobility component protein 13 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human high mobility component protein 13 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, etc.
  • Techniques for preparing monoclonal antibodies to human high mobility component protein 13 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). And existing techniques for producing single-chain antibodies (US Pat No. 4 946778) can also be used to produce single chain antibodies against human high mobility component protein 13.
  • Antibodies to human high mobility component protein 13 can be used in immunohistochemical techniques to detect human high mobility component protein 13 in biopsy specimens. Monoclonal antibodies that bind to human high mobility component protein 1 3 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human high mobility component protein 1 3 High affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human high mobility component proteins 1 3 Positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human high mobility component protein 1 3.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human high mobility component protein 1 3.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human high mobility component protein 13 levels.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of human high mobility component protein 1 3 detected in the test can be used to explain the importance of human high mobility component protein 1 3 in various diseases and to diagnose human high mobility component protein 13 Effect of disease.
  • 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 high mobility component protein 1 3 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human high mobility component protein 1 3.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human high mobility component protein 1 3 to inhibit endogenous human high mobility component protein 13 activity.
  • a mutated human high mobility component protein 1 3 may be a shortened human high mobility component protein 1 3 lacking a signaling functional domain. Although it can bind to a downstream substrate, it lacks signaling. active.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human high mobility component protein 1 3.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human high mobility component protein 1 3 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human high mobility component protein 13 can be found in the existing literature (Sambrook, et al.).
  • a polynucleotide encoding human high mobility component protein 13 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides that inhibit human high mobility component protein 1 3 mRNA and Ribozymes are also within the scope of the invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA and D 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 downstream of the vector's RNA 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 high mobility component protein 1 3 can be used for the diagnosis of diseases related to human high mobility component protein 1 3.
  • the polynucleotide encoding human high mobility component protein 1 3 can be used to detect the expression of human high mobility component protein 1 3 or the abnormal expression of human high mobility component protein 1 3 in a disease state.
  • the DNA sequence encoding human high mobility component protein 1 3 can be used to hybridize biopsy specimens to determine the expression of human high mobility component protein 1 3.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and the relevant kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microray) or a DNA chip (also known as a "gene chip"), and used to analyze differential expression analysis and gene diagnosis of genes in tissues.
  • a microarray Microray
  • a DNA chip also known as a "gene chip”
  • Human high mobility component protein U specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human high mobility component protein 1 3 transcription products.
  • Human high mobility component protein 1 3 mutations include point mutations, translocations, deletions, recombinations and any other abnormalities compared to the normal wild type human high mobility component protein 1 3 D sequence. Mutations can be detected using existing techniques such as Sou thern blotting, DNA 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.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, the specific loci of each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome locations. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 1-35 bp) are prepared based on cDNA, and the sequences can be mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain 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 by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific 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. Mckus i ck, Mende l ian Inher i tance in Man (available online with Johns Hopkins University Welch Medica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all 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 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 high mobility component protein 13 for effective treatment and / or prognosis Dosage for a specific indication.
  • the amount and dosage range of human high mobility component protein 13 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.
  • the sequence of the human high mobility component protein 13 gene and the encoded protein sequence of the present invention were analyzed by the Mas t program (Basicloca l Al ignment search tool) [Al tschul, SF et al. J. Mol. Biol. 1990 215: 403-10], perform homology search in databases such as Genbank and Swiss sport.
  • the gene with the highest homology to the human high mobility component protein 13 gene of the present invention is a known human HMG-2a gene, N00 / 00333
  • the accession number of the encoded protein in Genbank is Y10043.
  • the protein homology results are shown in Figure 1. The two are highly homologous, with 75% identity; 75% similarity.
  • Example 3 Cloning of Human High Mobility Component Protein 1 3 Gene 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. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Pr imer 1 5'-TCTATAAGGAGAGCTATGCTGCGT-3 '(SEQ ID NO: 3)
  • Pr imer2 5'-GACAAGGTCCTTCTCTGCCATCCA-3 '(SEQ IDNO: 4)
  • Pr imerl is a forward sequence located at the 5th end of bp in SEQ ID NO1; Pr imer2 is a 3 'end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 ⁇ l of KC1, 10mraol / L Tris-Cl, (pH 8.5.5), 1.5 mmol / L MgC12, 2 (mol / L dNTP) , l Opmol primer, 1U Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C, 30sec; 55 ° C, 30sec. 72 ° C, 2min.
  • RNA precipitate 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M acetic acid Sodium (pH4.0) was homogenized into the ground, and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol were added. 49: 1), mixed and centrifuged. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. Wash the obtained RNA precipitate with 70% ethanol, dry and dissolve in water.
  • RNA was electrophoresed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2 M formaldehyde. Then Preparation 32 ⁇ - DNA labeled probes were transferred to nitrocellulose membranes. cr 32 P dATP using random primers SYSTEM. the DNA probe used is shown in Figure 1 PCR amplification of human high mobility component proteins 1 3 coding sequence (858bp to 1226bp).
  • Primer 3 5'-CCCCATATGATGACCAACTCAGGGAGGCGAG-3 '(Seq ID No 5)
  • Primer4 5,-CCCGGATCCTCACAAAGAGACAGATAAAAC -3, (Seq ID No 6)
  • the 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences at the 5 and 3' ends of the target gene, respectively.
  • the BamHI restriction site corresponds to a selective endonuclease site on the expression vector plasmid pET-28b (+) (product of Novagen, Cat. No. 69865.3).
  • a PCR reaction was performed using the P BS-0309e03 plasmid containing the full-length target gene as a template.
  • PCR reaction conditions were: 1 in a total volume of 50 ⁇ plasmid pBS-0309e03 containing 10pg, Primer- 3 primer Primer-4, and j is the other points 10p so ol, Advantage polymerase Mix (Clontech Products) 1 ⁇ 1!. Cycle parameters: 94. C 20s, 60. C30s, 68. C 2 min, a total of 25 cycles. Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. Ligation products were transformed by the calcium chloride method A DH5 E.
  • coli bacteria after overnight containing kanamycin (final concentration of 30 ⁇ ⁇ / ⁇ 1) LB plates, screened by colony PCR and the positive clones and sequenced.
  • a positive clone (pET-0309e03) with a correct sequence was selected, and the recombinant plasmid was transformed into a large intestine rod BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • the host bacteria BL21 (pET-0309e03) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1mmol / 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.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following human high mobility component protein 13-specific peptides: NH 2 -Met-Thr-Asn-Ser-Gly-Arg-Arg-Glu-Asp-Val-Pro- Arg-Arg-Pro-Pro-COOH (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine HMG-13, 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 du léiomyome de l'utérus. L'invention concerne aussi l'agoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humaine HMG-13.
PCT/CN2000/000333 1999-10-18 2000-10-16 Nouveau polypeptide, une proteine humaine hmg-13, et polynucleotide codant pour ce polypeptide WO2001029080A1 (fr)

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AU78993/00A AU7899300A (en) 1999-10-18 2000-10-16 A novel polypeptide - human high mobility group protein 13 and a polynucleotide encoding the same

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19548122A1 (de) * 1995-12-21 1997-06-26 Joern Prof Dr Bullerdiek Nukleinsäuresequenzen von Genen der High Mobility Group Proteine sowie Verwendungen derselben

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19548122A1 (de) * 1995-12-21 1997-06-26 Joern Prof Dr Bullerdiek Nukleinsäuresequenzen von Genen der High Mobility Group Proteine sowie Verwendungen derselben

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
VACCARI T. ET AL.: "Hmg4, a new member of the Hmg/12 gene family", GENOMICS, vol. 49, no. 2, April 1998 (1998-04-01), pages 247 - 252 *
WILKE K. ET AL.: "Isolation of human and mouse HMG2a cDNAs: evidence for an HMG2a-specific 3' untranslated region", GENE, vol. 198, no. 1-2, October 1997 (1997-10-01), pages 269 - 274 *

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