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WO2001029076A1 - Nouveau polypeptide, une proteine-22 humaine p24, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, une proteine-22 humaine p24, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001029076A1
WO2001029076A1 PCT/CN2000/000332 CN0000332W WO0129076A1 WO 2001029076 A1 WO2001029076 A1 WO 2001029076A1 CN 0000332 W CN0000332 W CN 0000332W WO 0129076 A1 WO0129076 A1 WO 0129076A1
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Prior art keywords
polypeptide
protein
polynucleotide
human
seq
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PCT/CN2000/000332
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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 AU78992/00A priority Critical patent/AU7899200A/en
Publication of WO2001029076A1 publication Critical patent/WO2001029076A1/fr

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

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, human P24 protein -22, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • the P24 protein family consists of membrane integrins, all of which contain a C-terminal transmembrane domain. All P24 proteins have a C-terminal transmembrane phenylalanine residue in the cytoplasmic region. In all known P24 proteins, this conserved phenylalanine residue is immediately followed by 2-3 amino acid residues. The different arrangement of C-terminal fluorene residues determines that the P24 protein can bind to different subunits of the COP-coatmer complex.
  • the homologous protein yp24A (also known as Emp24p) of P24 in yeast has been isolated from COP coats derived from endoplasmic reticulum and is related to the efficient transport of a group of secreted proteins transported from the endoplasmic reticulum to the Golgi apparatus (Stamnes et al. al. EMBO J. 1995; 14: 1329-1339) 0 Electron microscopy of P24A-disabled yeast cells showed a continuous decrease in the accumulation of transport vesicles, indicating that yp24A is necessary for the formation of transport vesicles. Since only a portion of yeast secreted protein transport is affected in yp24A mutant cells, Schimoller et al.
  • P24 yeast homologs can recognize and capture completely different, or may be partially identical, secreted proteins into secretory vesicles.
  • Rothman et al. (1996; Science 272: 227-234) speculated that P24 homologues may be used as cargo receptors to select proteins for loading into COP coats and vesicles.
  • Rat p24A is abundantly expressed in the pancreas, which is consistent with its presumed role in the secretory pathway.
  • a protein that is significantly homologous to the p24 family was found in human glioma cell lines (Gayle, M. A. et al. (1996) J. Biol. Chem. 271: 5784-5789).
  • the protein can bind to the interleukin 1 receptor analog T1 / ST2, however, it does not show biological activity in the IL-1 or T1 / ST2 receptor-based assays. Therefore, the T1 / ST2 binding protein may be another member of the human P24 family.
  • 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 P24 protein-22. It is another object of the present invention to provide a genetically engineered host cell containing a polynucleotide encoding human P24 protein-22.
  • Another object of the present invention is to provide a method for producing human P24 protein-22.
  • Another object of the present invention is to provide antibodies against the polypeptide of the present invention-human P24 protein-22.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human P24 protein-22.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of human P24 protein-22. Summary of invention
  • a novel isolated human P24 protein-22 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 variant polypeptide thereof, or Active fragments, 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 92 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human P24 protein-22; (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 168-755 in SEQ ID NO: 1; and (b) a sequence having 1-2208 in SEQ ID NO: 1 Sequence of bits.
  • FIG. 1 is a comparison diagram of amino acid sequence homology between the human P24 protein -22 of the present invention and the mouse P24 protein.
  • the upper sequence is human P24 protein -22, and the lower sequence is mouse P24 protein.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • FIG. 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of isolated human P24 protein-22. 21. 4kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. Summary of the invention
  • 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 P24 protein -22 means that human P24 protein -22 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human P24 protein-22 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human P24 protein-22 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human P24 protein-22, 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 may be naturally purified products or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human P24 protein-22.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human P24 protein -22 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by the genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by other groups to include a substituent; or (in) such One, in which the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 2208 bases in length and its open reading frame (168-755) encodes 195 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide is highly homologous to the mouse P24 protein (D83206) with 91% identity. It can be inferred that the human P24 protein-22 has the mouse P24 protein phase. Similar structure and function.
  • the polynucleotide of the present invention may be in the D form or the RNA form.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • 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, 6 (TC; or (2) Add a denaturant 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
  • the homology between the two is at least 95%, and more preferably 97 »/» or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biology as the mature polypeptide shown in SEQ ID NO: 2 Function and activity.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, 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 P24 protein-22.
  • 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 P24 protein-22 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 DM 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) DM-DM or DNA-RNA hybridization; (2) the appearance or loss of marker gene function; (3) measuring the level of human P24 protein-22 transcripts; (4) Detection of gene-expressed protein products by immunological techniques or determination of biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually 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.
  • the D probe can be labeled with a radioisotope, luciferin, or an enzyme (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 of human P24 protein-22 gene expression.
  • ELISA enzyme-linked immunosorbent assay
  • a method for amplifying DNA / R using PCR technology is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid cDNA end rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various 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). Fixed. 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 produced by genetic engineering using the vector of the present invention or directly using a human P24 protein-22 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding the human P24 protein-22 may 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, etal.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing the D sequence encoding human P24 protein-22 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, e.t. a. Mo l ecu lar Cloning, a Labora tory Manua l, cod d 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 for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers from 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, and green for eukaryotic cell culture. 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, and green for eukaryotic cell culture.
  • GFP Fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human P24 protein-22 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or 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 Sf 9
  • animal cells such as CH0, COS or Bowes 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 absorbing DNA can be harvested after the exponential growth phase, and treated with the ( 12 method, the steps used are well known in the art.
  • MgC l 2 If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipid Body packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human P24 protein-22 (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 and the antagonists, agonists and inhibitors of the polypeptides can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and Immune diseases, etc.
  • Membrane-bound receptor and ion channel transport disorders and cystic fibrosis [cys ticf ibros is transmembrane conduc tance regula tor; CFTR], glucose-galactose malabsorption [Na. Sup. + / Glucose cotransporter], high jk months It is related to low-dens i ty li poprote in (LDL) receptor and diabetes [insul in receptor].
  • Abnormal hormone secretion can cause various disorders, such as diabetes insipidus [vasopres s in], hypoglycemia [insul in, g lucagon], Graves' sdi sease (exophthalmic goiter, common in women) And thyroid hormones. Cushing's disease (hyperadrenalism secondary to excessive secretion of adrenocortical hormones in the anterior pituitary gland) and Edison's disease (hyperadrenal function) [adrenocor t icotropic hormone; ACTH].
  • Benign tumor syndrome (abdominal colic, diarrhea, valve heart) caused by overdose Disease); ectopic synthesis and secretion of bioactive proteins such as ACTH and vasopressin in lung and pancreatic cancer, parathyroid hormone in lung and bladder cancer, calcitonin in lung and breast cancer, medullary thyroid tumor Thyroid-stimulating hormone.
  • New human HP24 polynucleotides that transport vesicle proteins provide a means to study vesicle transport and secretion under normal and pathological conditions.
  • the discovery of the new P24 vesicle transport protein meets the needs for the diagnosis and treatment of diseases associated with abnormal vesicle transport.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human P24 protein-22.
  • Agonists enhance biological functions such as human P24 protein -22 to stimulate 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 P24 protein -22 can be cultured with labeled human P24 protein -22 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human P24 protein-22 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human P24 protein -22 can bind to human P24 protein -22 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human P24 protein-22 When screening compounds as antagonists, human P24 protein-22 can be added to bioanalytical assays to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between human P24 protein-22 and its receptor. Using the same method for screening compounds as above, it can be screened to act as an antagonist Receptor deletions and analogs.
  • Polypeptide molecules capable of binding to human P24 protein-22 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the human P24 protein-22 molecule should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human P24 protein-22 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human P24 protein-22 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human P24 protein-22 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridization Tumor technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrison et al., PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against human P24 protein-22.
  • Antibodies against human P24 protein -22 can be used in immunohistochemical techniques to detect human P24 protein -22 in biopsy specimens.
  • Monoclonal antibodies that bind to human P24 protein -22 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 P24 protein-22 high affinity monoclonal antibody 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 P24 protein-22 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human P24 protein-22. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human P24 protein-22.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human P24 protein-22 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human P24 protein -22 detected in the test can be used to explain the importance of human P24 protein -22 in various diseases and to diagnose diseases in which human P24 protein -22 functions.
  • the polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding human P24 protein-22 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 P24 protein-22.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human P24 protein-22 to inhibit endogenous human P24 protein-22 activity.
  • a mutated human P24 protein-22 may be a shortened human P24 protein-22 that lacks a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human P24 protein-22.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human P24 protein-22 into cells.
  • recombinant viral vectors carrying a polynucleotide encoding human P24 protein-22 can be found in existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human P24 protein-22 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 P24 protein-22 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DM, and ribozymes can be obtained using any existing RNA or DNA synthesis techniques, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.
  • the polynucleotide encoding human P24 protein-22 can be used for the diagnosis of diseases related to human P24 protein-22.
  • the polynucleotide encoding human P24 protein-22 can be used to detect the expression of human P24 protein-22 or the abnormal expression of human P24 protein-22 in a disease state.
  • the DNA sequence encoding human P24 protein-22 can be used to hybridize biopsy specimens to determine the expression of human P24 protein-22.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human P24 protein-22 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human P24 protein-22 transcripts. Detection of mutations in the human P24 protein-22 gene can also be used to diagnose human P24 protein-22-related diseases.
  • Human P24 protein-22 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human P24 protein-22 DNA 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 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 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 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those 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. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Wetch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using 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 Mb mapping resolution and one gene per 20 kb).
  • 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 P24 protein-22 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human P24 protein-22 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 0492F02 cDNA sequence is new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the results showed that the full-length cDNA contained in the 0492F02 clone was 2208bp (as shown in SEQ ID NO: 1), and there was a 587bp open reading frame (0RF) from 168bp to 755bp, encoding a new protein (such as SEQ ID NO : Shown in 2).
  • This clone pBS-0492F02 the encoded protein Named human P24 protein-22.
  • Example 2 Homologous search of cDNA clones
  • CDNA was synthesized by reverse transcription reaction using fetal brain cell total R as a template and oligo-dT as a primer. After purification with Qiagene's kit, PCR was performed using the following primers:
  • Primerl 5 '-GGGTGGCCGACGGCGATGGGACCCCAG-3' (SEQ ID NO: 3)
  • Primer2 5 '-CTTGTTCACATATTTTATTTATCCAGTG-3' (SEQ ID NO: 4)
  • Primerl is located in the forward direction of the 5th end of SEQ ID NO: 1, starting at lbp Sequence
  • Primer 2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions A reaction volume of 50 ⁇ l contains 50 mmol / L KC1, 10 cryptool / L Tris-Cl, (pH8.5), 1.5 mmol / 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.
  • RT-PCR set ⁇ -act in as a positive control and template blank 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 1-2208bp shown in SEQ ID NO: 1.
  • RNA extraction in one step involves acid guanidinium thiocyanate phenol-chloroform extraction. 4M guanidine isothiocyanate-25 mM sodium citrate, 0.2M acetic acid Sodium (pH 4.0) was used to homogenize the tissue, 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) were added, and the mixture was centrifuged. The aqueous phase layer was aspirated, and isopropyl alcohol (0.8 (Volume) and the mixture was centrifuged to obtain RM pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7. 0) -5mM sodium acetate-ImM EDTA- 2.2M formaldehyde on 1.2% agarose gel Line electrophoresis. It was then transferred to a nitrocellulose membrane.
  • the DNA probe used was the PCR amplified human P24 protein-22 coding region sequence (168bp to 755bp) shown in FIG.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH 7.4) -5 x SSC-5 x Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, place the filter at 1 x SSC-0.1 ° /. Wash in SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Primer3 5'-CCCGGATCGATGAGTTCTTGCAGCAACGTCTGTG-3 '(Seq ID No: 5)
  • Primer 4 5'-CATGCGGCCGCCCGGTTGCCAGTAGAGGCTGGAC-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain BamHI and Notl restriction sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the restriction sites of BamHI and Notl correspond to the selectivity on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • a PCR reaction was performed using the PBS-0492F02 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of PBS-0492F02 plasmid, primers Primer-3 and Primer-4 were 10 pmol, and Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles.
  • the amplified product and plasmid pET-28 (+) were double digested with BamHI and Notl, respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E.
  • the polypeptide is coupled with 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. Rabbits were immunized with 4 mg of the hemocyanin-polypeptide complex plus complete Freund's adjuvant. After 15 days, the rabbit was immunized with hemocyanin polypeptide complex plus incomplete Freund's adjuvant once. ⁇ The titer of antibody in rabbit serum was measured by ELISA using a 15 g / ml bovine serum albumin peptide complex-coated titer plate. Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit sera.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to human P24 protein-22.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine-22 humaine P24, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'agoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la nouvelle protéine-22 humaine P24.
PCT/CN2000/000332 1999-10-18 2000-10-16 Nouveau polypeptide, une proteine-22 humaine p24, et polynucleotide codant pour ce polypeptide WO2001029076A1 (fr)

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AU78992/00A AU7899200A (en) 1999-10-18 2000-10-16 A novel polypeptide-human p24 protein-22 and the polynucleotide encoding said polypeptide

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CN99116990 1999-10-18
CN99116990.5 1999-10-18

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"A newly identified membrane protein localized exclusively in intracellular organelles of neurons", BRAIN RES. MOL., vol. 46, no. 1-2, 1997, pages 265 - 273 *

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