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WO2001029075A1 - Nouveau polypeptide, proteine 129 activant la proteine g, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine 129 activant la proteine g, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001029075A1
WO2001029075A1 PCT/CN2000/000329 CN0000329W WO0129075A1 WO 2001029075 A1 WO2001029075 A1 WO 2001029075A1 CN 0000329 W CN0000329 W CN 0000329W WO 0129075 A1 WO0129075 A1 WO 0129075A1
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Prior art keywords
protein
polypeptide
polynucleotide
sequence
seq
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PCT/CN2000/000329
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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 AU10156/01A priority Critical patent/AU1015601A/en
Publication of WO2001029075A1 publication Critical patent/WO2001029075A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C07K14/4706—Guanosine triphosphatase activating protein, GAP
    • 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, G protein activating protein 129, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide.
  • the G protein family can be divided into many subfamilies.
  • GAP can also be divided into many subfamilies, and each GAP specifically acts on a G protein.
  • Ro protein is a member of G protein family Ras. It includes CDC42 protein found in Streptomyces species and Rho, Rac and Cdc42Hs found in mammals. It plays an important role in nuclear skeletal tissue, signaling pathways, and early embryonic development. Studies have found that the protein encoded by the human breakpoint cluster region gene (bcr) has Rho protein activation. Its structural characteristics are as follows: There is a peptide composed of about 200 amino acid residues, forming an ⁇ -helix, which is called the RhoGAP domain. The human gene of the present invention has 37% homology with the nematode G protein activating protein at the protein level. It is very similar to the characteristic domain of the Rho protein activation protein subfamily ... the RhoGAP domain.
  • bcr human breakpoint cluster region gene
  • the novel gene of the present invention is considered to be a gene encoding a GAP protein similar to the nematode GAP protein, and is named human G protein activating protein 129. It is inferred that it is similar to RhoGAP domain and has similar biological functions.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a method for producing a G protein activating protein 129.
  • Another object of the present invention is to provide antibodies against the polypeptide G protein activating protein 129 of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the polypeptide G protein activating protein 129 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of G protein activating protein 129. Summary of invention
  • a novel isolated G protein activating protein 129 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 70 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the above-mentioned G protein activating protein 129; (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 518-4028 in SEQ ID NO: 1; and (b) a sequence having 1-4828 in SEQ ID NO: 1 Sequence of bits.
  • Fig. 1 is a comparison diagram of amino acid sequence homology between G protein activated protein 129 of the present invention and nematode G protein activated protein.
  • the upper sequence is G protein activating protein 129, and the lower sequence is nematode G protein activating protein.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated G protein-activated protein 129.
  • 129 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 G protein activated protein 129 means that G protein activated protein 129 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify G protein-activated protein 129 using standard protein purification techniques. Substantially pure peptide on non-reducing polyacrylamide gel Can produce a single main band. The purity of the G protein-activated protein 129 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, G protein activating protein 129, 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 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.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the starting methionine residue.
  • the present invention also includes fragments, derivatives and analogs of G protein activating protein 129.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the G-protein activated protein 129 of the present invention.
  • a fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a type in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution is The amino acid may or may not be encoded by a genetic codon; or (II) a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (I ⁇ ) Such a type in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a type in which an additional amino acid sequence is fused into a mature polypeptide and the polypeptide sequence (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) As set forth herein, such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the
  • 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 full-length polynucleotide sequence of 4828 bases and its open reading frame (518-4039) encodes 1173 amino acids.
  • the polypeptide has 37% homology with the G protein-activated protein of the nematode, and the polypeptide has a conserved base of the G protein Rho-activated protein subfamily, and the new human G protein can be inferred Activated proteins have similar structures and functions as the Rho activated protein subfamily.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the D form includes 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 that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (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 a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.
  • 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, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding G protein activating protein 129.
  • 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 G protein activating protein 129 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) separating a double-stranded DM sequence from genomic DNA; 2) chemically synthesizing a DNA sequence to obtain the double-stranded DM of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. Criteria for isolating cDNA of interest The method is to isolate mRNA from donor cells that highly express the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for mRNA extraction, 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 cDIv: libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determining the level of transcripts of G protein-activated protein 129; (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 2,000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of G protein activated protein 129 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method for amplifying DNA / RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a G protein activation protein 129 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .
  • a polynucleotide sequence encoding a G protein activating protein 129 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, 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 DM sequences encoding G protein activating protein 129 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecu lar Cl on ing, a Labora tory Manua l, cold Spin Harbor Labora tory. New York, 1989).
  • the Li 1 J bad sequence may be operably linked to an appropriate promoter in the expression vector, to direct mRNA synthesis. Representative examples of these promoters are: the l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for D expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a G protein activating protein 129 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 insect cells
  • insect cells such as flies 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 CaC 12 method, the steps used are well known in the art. Alternatively, M g C 12 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.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant protein G activating protein 129 (Science, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases. Because hGAP129 can be combined with Rho protein, Rho is inactivated by hydrolyzing GTP to GDP, so it can be used for treatment and prevention Cancers caused by Rho protein mutations, including but not limited to: gallbladder cancer, breast cancer, rectal cancer, kidney cancer, lung cancer, ovarian cancer, spleen cancer, gastric cancer, malignant melanoma, teratoma, neuroblastoma , Gliomas, and tumors of blood and body fluid origin.
  • the present invention can also be used to treat other diseases caused by Rho mutation, such as diseases of metabolic disorder, developmental disorders, and visual disorders.
  • the invention also provides screening compounds to identify improved (agonist) or repressed (antagonist) G protein activation Method of pharmacy of protein 129.
  • Agonists enhance biological functions such as G protein-activating protein 129 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing G protein activating protein 129 can be cultured together with labeled G protein activating protein 129 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of G protein activating protein 129 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of G protein activating protein 129 can bind to G protein activating protein 129 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.
  • G protein-activated protein 129 When screening compounds as antagonists, G protein-activated protein 129 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between G protein-activated protein 129 and its receptor. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to G protein activating protein 129 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, the G protein-activated protein 129 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 G protein activating protein 129 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 G protein-activated protein 129 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 G protein-activated protein 129 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.
  • the chimeric antibody variable region and a human constant region of non-human origin in combination produce the available prior art (Mor ri son etal, PNAS, 1985, 81: 6851) 0 only some technical production of single chain antibodies (US Pa t No. 4946778) can also be used to produce single-chain antibodies against G-protein activated protein 129.
  • Antibodies against G protein activating protein 129 can be used in immunohistochemical techniques to detect G protein activating protein 129 in biopsy specimens.
  • Monoclonal antibodies that bind to G protein activated protein 129 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.
  • G protein activated protein 129 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 G protein-activated protein 129-positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to G protein activating protein 129.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of G protein-activating protein 129.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of G protein-activated protein 129 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of G-protein-activated protein 129 detected in the test can be used to explain the importance of G-protein-activated protein 129 in various diseases and to diagnose diseases in which G-protein-activated protein 129 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.
  • G protein activating protein 129 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormalities in cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of G protein activating protein 129.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated G-protein-activating protein 129 to inhibit endogenous G-protein-activating protein 129 activity.
  • a mutated G-protein-activating protein 129 may be a shortened G-protein-activating protein 129 lacking a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of G protein activating protein 129.
  • Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, parvoviruses, and the like can be used to transfer polynucleotides encoding G protein activating protein 129 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a G protein activating protein 129 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding G protein activating protein 129 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit G protein activating protein 129 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA and DNA and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology of solid phase phosphate amide synthesis of oligonucleotides has been widely used.
  • Antisense RNA molecules can be expressed in vitro or Obtained in vivo transcription.
  • This D sequence has been integrated downstream of the vector's RNA polymerase promoter.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding G protein activating protein 129 can be used for the diagnosis of diseases related to G protein activating protein 129.
  • Polynucleotides encoding G protein activating protein 129 can be used to detect the expression of G protein activating protein 129 or the abnormal expression of G protein activating protein 129 in a disease state.
  • the DNA sequence encoding G protein activating protein 129 can be used to hybridize biopsy specimens to determine the expression of G protein activating protein 129.
  • Hybridization techniques include Sout hern blotting, Nor thern blotting, and in situ hybridization. These technical methods are all mature technologies that are publicly available, and related kits are commercially available.
  • Part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array (Mi croar ray) or a DM chip (also known as a "gene chip"), and used to analyze differential expression analysis and gene diagnosis of genes in tissues.
  • G-protein-activated protein 129-specific primers can also be used to detect G-protein-activated protein 129 transcription products by in vitro amplification of RNA-polymerase chain reaction (RT-PCR).
  • G protein activating protein 129 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type G protein activating protein 129 DNA sequence. Mutations can be detected using existing techniques such as Sou thern blotting, D 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 of a human chromosome and can hybridize with it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeat polymorphisms) are available for labeling chromosomal positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DM sequences on a chromosome.
  • the PCR primers (preferably 15-35bp) are prepared according to cD, and the sequences can be located on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DM 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 pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization (FI SH) of cDNA clones with metaphase chromosomes can be accurately performed in one step Chromosomal mapping.
  • FI SH 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, for example, in V. Mckus Ick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all of the affected individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them.
  • the polypeptide of the present invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • G protein activating protein 129 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of G-protein activating protein 129 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.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using QuikmRM Isolationist (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA Cloning Kit purchased from Clontech was used to insert the CDM fragments into the multicloning site of pBSK (+) vector (Clontech) to transform DH5 ⁇ to form a CDM library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and the cDNA sequence of one of the clones 0075d01 was found to be a new DM.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the 0075d01 clone contained a full-length cDNA of 4838bp (as shown in Seq ID NO: 1), and a 3522bp open reading frame (0RF) from 518bp to 4039bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone P BS-0075d01 was named G protein activating protein 129.
  • Example 2 Homologous search of cDNA clones
  • the sequence of the G protein activating protein 129 of the present invention and the protein sequence encoded by the G protein activating protein 129 were performed using the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] Genbank, Swissport and other databases perform homology search.
  • the gene with the highest homology to the G protein activating protein 129 of the present invention is a known nematode G protein activating protein gene, and its accession number to Genbank is 1102289.
  • the results of protein homology are shown in Figure 1. The two are highly homologous, with an identity of 37% and a similarity of 54%.
  • Example 3 Cloning of a gene encoding G protein activating protein 129 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer.
  • PCR amplification was performed with the following primers:
  • Primer 1 5'- ACGGCTGCGAGAGACGAAGCTTAGG-3 '(SEQ ID NO: 3)
  • Primer2 5'- TAAAACAATTTTTATTTCCAGTGT- 3, (SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer 2 is the 3 'terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l / L C1, 10mmol / L Tris- in a reaction volume of 50 ⁇ 1 CI, (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 1-4838bp shown in SEQ ID NO: 1.
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] rempliThis method involves acid guanidinium thiocyanate phenol-chloroform extraction. 4M guanidine isothiocyanate-25mM 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 an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA was used in a solution containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7. 0) -5mM sodium acetate - lmM EDTA- 2.2M electrophoresed on a 1.2% agarose formaldehyde gel and transferred to nitrocellulose with a- 32 P dATP labeled DM 32 ⁇ - prepared by the random primer method.
  • Probe The DM probe used is the sequence of the 129 coding region (518bp to 4039bp) of the G protein activated protein PCR amplified as shown in Figure 1.
  • Example 5 Recombination Expression, isolation and purification of G protein activated protein 129 in vitro
  • Primer3 5'- CCCGGATCCATGGAGGAAAGAAAAGCCTCGA-3 '(Seq ID No: 5)
  • Primer4 5'- CCCGCGGCCGCTTAAAGACAGGGATGAAGCTC-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ncol and BamHI digestion sites, respectively, followed by the coding sequences of the 5 'and 3' ends of the target gene, respectively.
  • Ncol and BamHI restriction sites correspond to selective endonuclease sites on the expression vector plasmid pET-8b (+) (Novagen, Cat. No. 69865.3).
  • the pBS-0075d01 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of P BS-0075d01 plasmid, primers Primer-3 and Primer- 4 points, and 1 J was lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, total 25 Cycles. Ncol and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed into coliform bacteria DH5 ⁇ using the calcium chloride method, and cultured overnight on LB plates containing kanamycin (final concentration 3 ( ⁇ g / ml)), and positive clones were screened by colony PCR method and sequenced. Positive sequence correct clone (pET-0075d01) was used to transform the recombinant plasmid into E. coli BL21 (DE3) plySs (product of Novagen) by calcium chloride method.
  • a peptide synthesizer (product of PE company) was used to synthesize the following G protein-activated protein 129-specific peptides: NH2-Met-G 1 uG 1 u-Arg-Lys-A 1 a—Ser— Ser- Thr- Ser- Pro- Pr oG 1 y-Asp-Ser-COOH (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. ⁇ Using a 15 g / ml bovine serum albumin peptide complex-coated titer plate as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The polypeptide bound to cyanogen bromide activated Sepharose4B column, by affinity chromatography from total i g G isolated anti-polypeptide antibody. The immunoprecipitation method proved that the purified antibody could specifically bind to G protein activated protein 129.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine 129 activant la protéine G, 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 maladies immunitaires, de troubles métaboliques, d'hypogenèse et de troubles visuels. L'invention concerne aussi l'agoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine 129 activant la protéine G.
PCT/CN2000/000329 1999-10-18 2000-10-16 Nouveau polypeptide, proteine 129 activant la proteine g, et polynucleotide codant pour ce polypeptide WO2001029075A1 (fr)

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AU10156/01A AU1015601A (en) 1999-10-18 2000-10-16 A novel polypeptide-g-protein activating protein 129 and the polynucleotide encoding the polypeptide

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CN99116986 1999-10-18
CN99116986.7 1999-10-18

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BHATTACHARYA D. ET AL.: "Molecular phylogenetic analysis of actin genic regions from achly bisexualis (oomycota) and costaria costata (chromophyta)", J. MOL. EVOL., vol. 33, no. 6, 1991, pages 525 - 536 *

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