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WO2001029077A1 - NOUVEAU POLYPEPTIDE, FACTEUR REGULATEUR HUMAIN 34 DE LA β 2-MICROGLOBULINE, ET POLYNUCLEOTIDE CODANT POUR CE POLYPEPTIDE - Google Patents

NOUVEAU POLYPEPTIDE, FACTEUR REGULATEUR HUMAIN 34 DE LA β 2-MICROGLOBULINE, ET POLYNUCLEOTIDE CODANT POUR CE POLYPEPTIDE Download PDF

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Publication number
WO2001029077A1
WO2001029077A1 PCT/CN2000/000334 CN0000334W WO0129077A1 WO 2001029077 A1 WO2001029077 A1 WO 2001029077A1 CN 0000334 W CN0000334 W CN 0000334W WO 0129077 A1 WO0129077 A1 WO 0129077A1
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WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
human
microglobulin
seq
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PCT/CN2000/000334
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Bioroad Gene Development Ltd
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Shanghai Bioroad Gene Development Ltd
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Priority to AU10157/01A priority Critical patent/AU1015701A/en
Publication of WO2001029077A1 publication Critical patent/WO2001029077A1/fr
Anticipated expiration legal-status Critical
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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 ⁇ 2 microglobulin transcription regulation factor 34, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
  • Gene expression plays an important regulatory role (Differentiation, 51: 201-207). At the same time, this gene may also be a reverse transcript, because its genetic structure has two short forward repeats, a polyadenylation tail signal and a polyadenylate tail (Mamm Genome, 9 (2 ): 103-106).
  • Class I major histocompatibility antigens and human ⁇ 2 microglobulin are non-covalently linked on the cell membrane, and play an important role in T-cell antigen recognition (Advance in Immunology, 27: 51-177), and allogeneic Or immune rejection during xenotransplantation is also closely related.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human P2 microglobulin transcriptional regulator 34.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding human P2 microglobulin transcriptional regulator 34.
  • Another object of the present invention is to provide a method for producing human ⁇ 2 microglobulin transcriptional regulator 34. Another object of the present invention is to provide an antibody against the polypeptide-human P2 microglobulin transcriptional regulatory factor 34 of the present invention.
  • Another object of the present invention is to provide a human microglobulin transcription regulation directed to the polypeptide of the present invention.
  • Section factor 34 mimics compounds, antagonists, agonists, and inhibitors.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human P 2 microglobulin transcription regulator 34.
  • a novel isolated human ⁇ 2 microglobulin transcription regulator 34 is provided.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2 or a conservative variation thereof Polypeptides, or active fragments thereof, or active derivatives, analogs thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 95 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human P 2 microglobulin transcription regulator 34; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID D NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 18 to 944 in SEQ ID NO: 1; and (b) a sequence having positions 1-1 in SEQ ID NO: 1 1 76-bit sequence.
  • Figure 1 is a comparison diagram of the amino acid sequence homology of the microglobulin transcription regulator 34 and the transcription regulator HCNGP in rat spleen tissue.
  • the upper sequence is human ⁇ 2 microglobulin transcription regulator 34,
  • the sequence below is the transcription regulating protein HCNGP in rat spleen tissue.
  • the same amino acid is represented by a single character amino acid between the two sequences, and the similar amino acid is represented by "+”.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human P 2 microglobulin transcription regulator 34. 33.9 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 existing in the natural state.
  • isolated human P 2 microglobulin transcription regulator 34 means that human P 2 microglobulin transcription regulator 34 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 P 2 microglobulin transcriptional regulatory factor 34 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 microglobulin transcription regulator 34 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human P 2 microglobulin transcription regulation factor 34, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptides of the invention may be glycosylated, or they may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of human ⁇ 2 microglobulin transcriptional regulator 34.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human P 2 microglobulin transcriptional regulator 34 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 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 Species, wherein the mature polypeptide is fused with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) a 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)
  • an additional amino acid sequence is fused into a mature polypeptide (such as Leader sequences or secreted sequences or sequences used to purify this
  • 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 CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 1176 bases in length and its open reading frame (18-944) encodes 308 amino acids.
  • this polypeptide is 94% homologous to the above-mentioned transcription regulator protein HCNGP in the spleen of the rat. It can be concluded that the new human microglobulin transcription regulator 34 has the transcription regulator protein HCNGP Similar structure and function.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, Due to DM or synthetic DNA.
  • DM can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the present invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Fi co ll, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 More than nucleotides.
  • Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human P 2 microglobulin transcriptional regulator 34.
  • 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 ⁇ 2 microglobulin transcription regulatory factor 34 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These technologies include However, it is not limited to: 1) hybridizing probes to a genomic or cDNA library to detect homologous polynucleotide sequences, and 2) antibody screening of the expression library to detect cloned polynucleotide fragments with common structural characteristics.
  • 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 D-sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the 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.
  • Various methods have been used to extract 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): (1) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the transcript of human P 2 microglobulin transcriptional regulator 34 Level; (4) detecting protein products 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.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human P 2 microglobulin transcriptional regulator 34 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).
  • 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 / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • Polynucleotide sequences of the gene of the present invention obtained as described above, or various DNA fragments can be used It is determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using the human ⁇ 2 microglobulin transcription regulator 34 coding sequence, and that the present invention is produced by recombinant technology Said method of polypeptide.
  • a polynucleotide sequence encoding human P2 microglobulin transcription regulator 34 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 (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 expression vectors containing a DNA sequence encoding human ⁇ 2 microglobulin transcriptional regulator 34 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DM technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an 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 for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells.
  • Enhancers are cis-acting factors expressed by DM, 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 and adenovirus enhancers on the late side of the origin of replication.
  • 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 for eukaryotic cell culture. And green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance for eukaryotic cell culture.
  • GFP green fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human microglobulin transcription regulatory factor 34 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell 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: 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 absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human ⁇ 2 microglobulin transcription regulator 34 (Sc ience, 1984; 224: 1431). Generally speaking, there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be 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.
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can treat cancer, cardiovascular diseases, neurological diseases, immune diseases, inflammation and the like.
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) human P 2 microglobulin transcriptional regulator 34.
  • Agonists enhance human ⁇ 2 microglobulin transcriptional regulator 34 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human P 2 microglobulin transcription regulator 34 can be cultured with labeled human P 2 microglobulin transcription regulator 34 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human microglobulin transcription regulator 34 include screened antibodies, compounds, receptor deletions, and the like. Antagonists of human P 2 microglobulin transcriptional regulator 34 can bind to human P 2 microglobulin transcriptional regulator 34 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide such that The polypeptide cannot perform biological functions.
  • human P 2 microglobulin transcription regulator 34 When screening compounds as antagonists, human P 2 microglobulin transcription regulator 34 can be added to the bioanalytical assay, and the interaction between human P 2 microglobulin transcription regulator 34 and its receptor can be determined by determining the compound Influence to determine if a compound 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 ⁇ 2 microglobulin transcription regulator 34 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, human ⁇ 2 microglobulin transcription regulator 34 molecules 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 against human ⁇ 2 microglobulin transcriptional regulator 34 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human P 2 microglobulin transcription regulator 34 directly into immunized animals
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human P 2 microglobulin transcription regulator 34 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-
  • Antibodies against human ⁇ 2 microglobulin transcription regulator 34 can be used in immunohistochemical techniques to detect human p 2 microglobulin transcription regulator 34 in biopsy specimens.
  • Monoclonal antibodies that bind to human P 2 microglobulin transcription regulator 34 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.
  • Such as human ⁇ 2 microglobulin transcriptional regulator 34 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 P 2 microglobulin transcription regulators 34 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human P 2 microglobulin transcriptional regulator 34.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human P 2 microglobulin transcriptional regulator 34.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human P 2 microglobulin transcriptional regulator 34.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human P 2 microglobulin transcription regulator 34 detected in the test can be used to explain the importance of human ⁇ 2 microglobulin transcription regulator 34 in various diseases and to diagnose human ⁇ 2 microglobulin transcription Diseases where regulator 34 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.
  • the polynucleotide encoding human P 2 microglobulin transcriptional regulator 34 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 microglobulin transcription regulator 34.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human ⁇ 2 microglobulin transcription regulator 34 to inhibit endogenous human ⁇ 2 microglobulin transcription regulator 34 activity.
  • a variant human ⁇ 2 microglobulin transcriptional regulator 34 may be a shortened human ⁇ 2 microglobulin transcriptional regulator 34 that lacks a signaling domain. Although it can bind to downstream substrates, it lacks Signaling activity.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human ⁇ 2 microglobulin transcription regulator 34.
  • 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 P 2 microglobulin transcription regulator 34 into a cell. Construction of a polynucleotide carrying a polynucleotide encoding human P 2 microglobulin transcription regulator 34 Methods for grouping viral vectors can be found in the existing literature (Sambrook, eta l.). In addition, a recombinant polynucleotide encoding human ⁇ 2 microglobulin transcription regulator 34 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human ⁇ 2 microglobulin transcription regulator 34 raRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the R. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human P 2 microglobulin transcription regulator 34 can be used for the diagnosis of diseases related to human ⁇ 2 microglobulin transcription regulator 34.
  • the polynucleotide encoding human P 2 microglobulin transcription regulator 34 can be used to detect the expression of human ⁇ 2 microglobulin transcription regulator 34 or the abnormal expression of human P 2 microglobulin transcription regulator 34 in a disease state.
  • a DNA sequence encoding human P 2 microglobulin transcription regulatory factor 34 can be used to hybridize biopsy specimens to determine the expression of human ⁇ 2 microglobulin transcription regulatory factor 34.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, in situ hybridization, and the like.
  • polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human ⁇ 2 microglobulin transcription regulator 34 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect the transcription products of human p 2 microglobulin transcription regulator 34.
  • Human ⁇ 2 microglobulin transcription regulator 34 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human ⁇ 2 microglobulin transcription regulator 34 DNA sequence. 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 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 Human chromosomes are in specific locations and can hybridize to them.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for labeling chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. 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 cD 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 ck, Mende l ian Inher i tance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the 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 changes in scabs 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 present invention also provides a kit or kit containing one or more containers, the containers containing one or more An ingredient of the pharmaceutical composition of the present invention.
  • the containers containing one or more An ingredient of the pharmaceutical composition of the present invention.
  • there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders authorize them to be administered to humans by government agencies that manufacture, use, or sell them.
  • 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 P 2 microglobulin transcription regulator 34 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human P 2 microglobulin transcription regulator 34 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 P 2 microglobulin transcription regulator 34 and the protein sequence encoded by the invention are identical to the sequence of the human P 2 microglobulin transcription regulator 34 and the protein sequence encoded by the invention.
  • CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction.
  • PCR amplification was performed with the following primers:
  • Primerl 5'-GGGGCTGTGGGAATAAGATGG-3 '(SEQ ID NO: 3)
  • Primer2 5'-AATACGGCACTTTTAATAGGCGG-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 ⁇ l of reaction volume in 50 ⁇ l containing 50 ol / L KC1, 10 ⁇ l / L Tris-Cl, (pH8.5), 1.5mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72. C 2min.
  • ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 1176 bp shown in SEQ ID NO: 1.
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159].
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue was homogenized with 4M guanidine isothiocyanate-25raM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) were added. ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA was electrophoresis was performed on a 1. agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-lmM EDTA- 2 . 2 M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation 32 P- DNA probe labeled with ot- 32 P dATP by random priming method. The D probe used was the PCR amplified human microglobulin transcription regulator 34 coding region sequence (18bp to 944bp) shown in FIG. 1.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4
  • Pr imer3 5'-CACCATATGATGGCGGGGAAGAAGAATGTTCTG-3 '(Seq ID No: 5)
  • Pr imer4 5,-CCCGGATCCTCACTGCTTGGCCTTCTTCAC-3' (Seq ID No: 6)
  • These two primers contain Ndel and Hindl ll digestion respectively Site, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively, and the Ndel and Hindl11 digestion sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3 Selective endonuclease sites on).
  • PCR was performed using the pBS-0165h06 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-0165h06 plasmid in a total volume of 50 ⁇ 1, Primer-3 and Primer-4 primers were 10 pmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Ndel and Hindl11 were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed into E.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following human ⁇ 2 microglobulin transcription regulator 34-specific peptides:
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avrameas, et al. Immunochemi s try, 1969; 6: 43. Use 4mg of the above jk cyanoprotein Peptide complex plus complete Freund's adjuvant was used to immunize rabbits. After 15 days, hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • HHCNGP antisense oligonucleotides inhibit proliferation of HL-60 cells
  • AS-0DN Anti-sense oligonucleotides
  • S-0DN sense oligonucleotides
  • TETD Tetraethylthiuram disulfide
  • AS-PS-0DN 5 '-GACTCGGGCTCTGAATCTTCCG-3' (SEQ ID NO.8)
  • S-PS-ODN 5'-CGGAAGATTCAGAGCCCGAGTC-3 '(SEQ ID NO.9)
  • HL-60 cells at a concentration of 5 ⁇ 10 5 were divided into 3 groups at 37.
  • AS-PS-ODN ⁇ S-PS-ODN final concentration is 20 g / ml.
  • Cell death was measured by trypan blue staining after 24 hours of cell culture. The results showed that the cells in the AS-PS-0DN group died The rate was 92.5%, and the mortality rate in the control and S-PS-0DN groups was 5%. This indicates that the antisense oligonucleotide of the HHCNGP gene can effectively inhibit the proliferation of HL-60 cells.
  • the present invention may be implemented by methods different from those specifically described above.

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Abstract

L'invention concerne un nouveau polypeptide, un facteur régulateur humain 34 de la β 2-microglobuline, 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 de cancers, de maladies cardio-vasculaires, de troubles du système nerveux, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'agoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour le facteur régulateur humain 34 de la β 2-microglobuline.
PCT/CN2000/000334 1999-10-18 2000-10-16 NOUVEAU POLYPEPTIDE, FACTEUR REGULATEUR HUMAIN 34 DE LA β 2-MICROGLOBULINE, ET POLYNUCLEOTIDE CODANT POUR CE POLYPEPTIDE Ceased WO2001029077A1 (fr)

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AU10157/01A AU1015701A (en) 1999-10-18 2000-10-16 A novel polypeptide-human beta 2 microglobin regulatory factor 34 and the polynucleotide encoding said polypeptide

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CN99116994 1999-10-18
CN99116994.8 1999-10-18

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WO2001029077A1 true WO2001029077A1 (fr) 2001-04-26

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PCT/CN2000/000334 Ceased WO2001029077A1 (fr) 1999-10-18 2000-10-16 NOUVEAU POLYPEPTIDE, FACTEUR REGULATEUR HUMAIN 34 DE LA β 2-MICROGLOBULINE, ET POLYNUCLEOTIDE CODANT POUR CE POLYPEPTIDE

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PALMER D.B. ET AL.: "The chromatin structure of the mouse beta-2-microglobulin locus", DIFFERENTIATION, vol. 51, no. 3, 1992, pages 201 - 207 *

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