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WO2002004504A1 - Nouveau polypeptide, sous-unite humaine 9.46 de proteine g, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, sous-unite humaine 9.46 de proteine g, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002004504A1
WO2002004504A1 PCT/CN2001/000985 CN0100985W WO0204504A1 WO 2002004504 A1 WO2002004504 A1 WO 2002004504A1 CN 0100985 W CN0100985 W CN 0100985W WO 0204504 A1 WO0204504 A1 WO 0204504A1
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Prior art keywords
polypeptide
human
polynucleotide
protein subunit
protein
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Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU93622/01A priority Critical patent/AU9362201A/en
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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

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide—a human G protein subunit 9.46, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • the evolution of organisms depends on the communication between cells. Due to the delicate division of labor among cells, some cell populations depend on other cell populations and require other cell populations to respond. This delicate intercellular communication network can control cell growth, division, death, differentiation to form tissues, and various life processes.
  • Hydrophilic signal molecules include neurotransmitters, growth factors, cytokines, local chemical transmitters, and most hormones. They cannot pass through the plasma membrane and can only bind to receptors on the cell surface to form ligand-receptor complexes for signaling. divert. According to the mechanism of signal transduction and the types of receptor proteins, cell surface receptors can be divided into three types: (1) ion channel-coupled receptors; (2) ligase surface receptors; (3) with G proteins Coupling receptor.
  • the receptor coupled to the G protein is a polypeptide surface receptor that crosses the plasma membrane seven times.
  • the binding of the ligand to the receptor changes the conformation of the receptor, allowing it to bind to the trimer of GTP-bound regulatory protein (G protein) on the inner side of the plasma membrane, and together activate the target protein (enzyme or ion channel) on the plasma membrane. ).
  • G protein GTP-bound regulatory protein
  • the ligand receptor complex acts on the effector indirectly through the G protein. Ion channels change the permeability of the plasma membrane to ions, and enzymes affect the behavior of cells by affecting intracellular substrates and other proteins.
  • the G protein couples the receptor and adenylyl cyclase, and converts extracellular signals into intracellular signals, that is, the second messenger of cAMP. Therefore, G protein is also called a coupling protein or a signal conversion protein. G protein consists of ⁇ , ⁇ and ⁇ subunits.
  • G protein has a wide range of functions in the body, involving important biological functions such as cell proliferation, signal transduction, protein synthesis, and protein localization.
  • GTP-binding proteins can be divided into various superfamilies, such as: Ras family, Rab family, etc. Each superfamily has many family members.
  • the protein synthesis elongation factor EF-Tu was the first G protein to be discovered. Later, people cloned a large number of other members of the GTP-binding protein family. member.
  • Members of the G protein family all contain a conserved GTP binding motif.
  • the GTP binding motif is used as a molecular switch in the body, and regulates the expression and non-expression of the protein through its binding to GDP and GTP. Therefore, G protein has a very important role in the body. Abnormal expression of G protein will lead to abnormal proliferation of tissue cells and abnormal expression of proteins, which will cause various related diseases, such as various malignant tumors and cancers, various Developmental disorders, various immune system diseases, etc.
  • the human G protein subunit 9.46 protein plays an important role in regulating important functions of the body, such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need to identify more participation in the field. These processes of the human G protein subunit 9.46 protein, in particular, identify the amino acid sequence of this protein. Newcomer G protein subunits 9. 46 The isolation of protein-coding genes also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human G protein subunit 9.46. '
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human G protein subunit 9.46.
  • Another object of the present invention is to provide a method for producing a human G protein subunit 9.46.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human G protein subunit 9.46.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1090 to 1350 in SEQ ID NO: 1; and (b) a sequence having 1-2034 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of the human G protein subunit 9.46 protein, which comprises utilizing the polypeptide of the invention.
  • the present invention also relates to a method for obtaining a disease or disease susceptibility associated with abnormal expression of the human G protein subunit 9.46 protein in vitro by using the method, which comprises detecting the polypeptide or a multi-core encoding thereof in a biological sample. Mutations in the nucleotide sequence, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention for the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human G protein subunit 9.46.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and can also refer to genomic or synthetic DNA or RNA, which can be single-stranded or double-stranded, representing the sense strand or Antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with the human G protein subunit 9.46, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds to the human G protein subunit 9.46.
  • Antagonist refers to a molecule that can block or modulate the biological or immunological activity of the human G protein subunit 9.46 when combined with the human G protein subunit 9.46.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds to the human G protein subunit 9.46.
  • Regular refers to a change in the function of human G protein subunit 9.46, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties, functions, or immunity of human G protein subunit 9.46 Change of nature.
  • substantially pure "means substantially free of naturally associated with other proteins, lipids, carbohydrates or other materials can be purified by standard protein purification techniques human G protein subunits 9.46.
  • the substantially pure human G protein subunit 9.46 can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human G protein subunit 9. 46 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to a polynucleotide that naturally binds by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T-G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences based on different methods, such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method groups each group by checking the distance between all pairs. The sequences are arranged in clusters. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A The number of spacer residues in a sequence B can also be determined by Clus ter method or using methods known in the art such as Jotun Hein.
  • the percent identity between nucleic acid sequences (Hein J., (1990) Methods in emzumology 183: 625-645) 0 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives encode major organisms that retain natural molecules Peptides with chemical properties.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to the human G protein subunit 9.46 epitope.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human G protein subunit 9. 46 means human G protein subunit 9. 46 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify the human G protein subunit 9.46 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 G protein subunit 9. 46 polypeptide can be analyzed by amino acid sequences.
  • the present invention provides a new polypeptide, a human G protein subunit 9.46, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of the human G protein subunit 9.46.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human G protein subunit 9.46 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 ( ⁇ ) such a type in which one or more amino acid residues A group on a base is substituted by another group to include a substituent; or (III) 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 polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) formed by fusing an additional amino acid sequence into a mature polypeptide.
  • such fragments, derivatives Analogs 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 full-length polynucleotide sequence of 2034 bases, and its open reading frame of 1090-1 350 encodes 86 amino acids.
  • this polypeptide has a similar expression profile with the human G protein ⁇ 4 subunit, and it can be inferred that the human G protein ⁇ 4 subunit has a similar function to the human G protein ⁇ 4 subunit.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DM can be a coding chain or a non-coding chain.
  • the coding region sequence encoding the 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 in the present invention, but which differs from the coding region sequence shown in SEQ ID NO: 1.
  • 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 is meant to include polynucleotides that encode such polypeptides and polynucleotides that include additional coding and / or noncoding sequences.
  • the present 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 present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • "strict conditions” means: (1) in the lower Hybridization and elution at ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) adding a denaturant such as 50% (v / v) formamide during hybridization, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only when the identity between the two sequences is at least 95%, and more preferably 97% or more Crosses occur.
  • 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 cores. 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 the human G protein subunit 9.46.
  • 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 G protein subunit 9.46 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • 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 developed for mRNA extraction, and kits are also commercially available (Q i agene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecula Cloning, A Laboratory Manua, Cold Harbor Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) DNA-DNA or DNA-RNA hybrids; (2) the presence or absence of marker gene functions; (3) determining the level of the human G protein subunit 9.46 transcript; (4) Detecting the protein product of gene expression by immunological technology or measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2,000 nucleotides, preferably within 1 000 nucleotides.
  • the probes used here are usually the gene sequence information of the present invention Based on the chemically synthesized DM sequence. The genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of the 9.46 gene expression of the human G protein subunit can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • the RACE method RACE-cDM terminal rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein.
  • 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 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 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 G protein subunit 9.46 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding a human G protein subunit 9.46 can be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, 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 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 a DNA sequence encoding the human G protein subunit 9.46 and appropriate transcription / translation regulatory elements. These methods include in vitro recombination DM technology, DNA synthesis technology, in vivo recombination technology, etc. (Sarabroook, et al. Molecular Cloning, a Laboratory Manua, Cold Spooning Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide niRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known controllable genes in prokaryotic or eukaryotic cells or A promoter expressed in its virus.
  • 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 expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription.
  • Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a human G protein subunit 9.46 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transforming a host cell with the Mia sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with (1 2 method used in the step are well known in the art. Alternatively, it is a MgCl 2. If If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging Wait.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human G protein subunit 9. 46 (Science, 1984; 224: 1431). Generally there are the following steps: (1). Use the polynucleotide (or variant) encoding the human human G protein subunit 9.46 of the present invention, or transform or transduce with a recombinant expression vector containing the polynucleotide A suitable host cell;
  • the medium used in the culture may be selected from various Conventional medium. 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 separated 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.
  • 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
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the human G protein subunit 9.46 and human G protein ⁇ 4 subunit of the present invention.
  • the upper graph is a graph of the expression profile of the human G protein subunit 9. 46
  • the lower graph is the graph of the expression profile of the human G protein ⁇ 4 subunit.
  • FIG. 2 is a polyacrylamide gel electrophoresis diagram (SDS-PAGE) of an isolated human G protein subunit 9.46. 9kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • 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 Quik mRNA I sola ti on Kit (product of Qiegene). 2ug po ly (A) mRNA is reverse transcribed CDNA is formed.
  • the Smart cDNA cloning kit purchased from Clontech
  • the bacteria formed a cDNA library.
  • Dye terminate cycle reaction sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones, 0502f08, was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5'— TACATATATCGCAATATGAAAATA —3, (SEQ ID NO: 3)
  • Primer2 5'- TTTACAGAATTTTTATTGTAAACA -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 'end of SEQ ID NO: 1;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 ⁇ l of Kol1, KC1, 10 ⁇ l / L Tris-CI, (pH8.5), 1.5mraol / L MgCl 2 , 200 ⁇ mol / L dNTP in a reaction volume of 50 ⁇ 1 , lOpmol primer, 1U Taq DNA polymerase (C 1 on t ech).
  • 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 the 1-2034bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human G protein subunit 9.46 gene expression:
  • RNA extraction in one step involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • the 32P- labeled probe (about 2x l0 6 cpm / ml) and transferred RM-nitrocellulose membrane was hybridized overnight at 42 ° C in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7.4) -5 SSC-5 ⁇ Denhardt's solution and 200 g / ml salmon Sperm DNA. After hybridization, wash the filter in 1 x SSC-0.1 ° / .SDS for 30 min at 55 ° C. Then, analyze and quantify using Phosphor Imager.
  • Example 4 In vitro expression, isolation and purification of recombinant human G protein subunit 9.46
  • Primer3 5 '-CCCCATATGATGGAATCTGGCTCTGTCTCCAGG-3' (Seq ID No: 5)
  • Primer4 5'-CATGGATCCTCACACCTGTAATCCCAGCACTTT-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5 'and 3' ends of the target gene, respectively.
  • the Ndel and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3).
  • the pBS-0502f08 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0502f 08 plasmid, primers Primer-3 and Primer-4 points; and “J” was lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E. coli DH5a by the calcium chloride method.
  • the bacteria were collected by centrifugation, and the supernatant was collected by ultrasonication. The supernatant was collected by centrifugation. Chromatography was performed using an affinity column His. Bind Quick Cartridge Novagen, which can bind 6 histidines (6His-Tag). The purified human protein G protein subunit was 9.46. After SDS-PAGE electrophoresis, a single band was obtained at 9 kDa ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following specific peptide of human G protein subunit 9.46: NH2- Met- Glu- Ser- Gly- Ser- Val- Ser- Arg- Leu- Glu- Cys- Ser- Gly- Ala- lie- COOH (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this example is to select a suitable oligonucleoside from the polynucleotide SEQ ID NO: 1 of the present invention.
  • the acid fragment is used as a hybridization probe, and the membrane hybridization method is used to identify whether some tissues contain the multinucleus of the present invention.
  • Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred size of the probe ranges from 18 to 50 nucleotides; 2.
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other unknown genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, see the references DeR isi, JL, Lyer, V. & Brown, P. 0. (1997) Science 278 ; 680-686. Schema, M., Cha i, A., Sha lom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between the points is 280 ⁇ ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips. The specific method steps have been variously reported in the literature. The post-spot processing steps of this embodiment are:
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are fetal brain, bladder mucosa, Ecv304 cell line of PMA +, thymus of Ecv304 cell line of LPS +, normal fibroblasts 1024NC, Fibroblast, growth factor stimulation, 1024NT, scar into fc Growth factor stimulation, 1013HT, scar into fc without growth factor stimulation, 1 G13HC, bladder cancer plant cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, Cardiac cancer. Draw a graph based on these 18 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profiles of the human G protein subunit 9.46 and human G protein ⁇ 4 subunit according to the present invention are very similar. Industrial applicability
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • the G protein-coupled receptor is a polypeptide surface receptor that crosses the plasma membrane seven times.
  • the binding of the ligand to the receptor changes the conformation of the receptor so that it binds to the regulatory protein (G protein) on the inside of the plasma membrane cell, and together activates the target protein (enzyme or ion channel) on the plasma membrane.
  • G protein regulatory protein
  • Ion channels change the permeability of the plasma membrane to ions, and enzymes affect cellular behavior by affecting intracellular substrates and other proteins.
  • Adrenaline receptors Adrenaline receptors, acetylcholine receptors, opioid receptors, glucagon receptors, insulin receptors, luteinizing hormone receptors, follicle stimulating hormone receptors, and thyroid stimulating hormone are known to belong to this class of receptors.
  • Receptors adrenocorticotropic hormone receptors, enterokinin receptors, and growth hormone release inhibitor factor receptors, etc. The dysfunction of these receptors can affect the physiological effects of the corresponding hormones, leading to various The occurrence of illness.
  • G protein consists of ⁇ , ⁇ and ⁇ subunits.
  • the human G protein subunit is a component of G protein. Abnormal expression in the body can affect the formation of G protein, cause G protein dysfunction, affect the physiological effects of corresponding ligands (such as hormones), and lead to the related diseases. occur.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human G protein subunit protein, and both have similar biological functions.
  • the polypeptide of the present invention is a component of the G protein in the body, and abnormal expression in the body can affect the formation of the G protein, cause dysfunction of the G protein, affect the physiological effects of the corresponding ligands (such as hormones), and then cause related diseases. Of these diseases, including but not limited to:
  • Adrenergic receptor acetylcholine receptor instability related diseases
  • arrhythmias such as atrial early, ventricular early, sinus tachycardia, supraventricular tachycardia, ventricular tachycardia, atrial flutter, atrial fibrillation, sinus bradycardia, sinus arrest, sick sinus syndrome, indoor conduction block, etc .;
  • CAD angina pectoris, myocardial infarction, cardiovascular neurosis, acute heart failure, chronic heart failure, ⁇ BP, neurogenic orthostatic hypotension, syncope, cerebrovascular accident, hypotension shock, etc .;
  • Pulmonary edema respiratory muscle paralysis, respiratory failure, bronchial asthma, etc .
  • Gastrointestinal neurosis Hydatid disease, psychogenic vomiting, nervousness, anorexia nervosa, irritable bowel syndrome, etc .;
  • Opioid peptide receptors are widely and unevenly distributed in the brain. Sites with high receptor density, such as the spinal glia, the medial thalamus, the ventricle, and the gray matter around the aqueduct, are all related to the introduction of painful stimuli, the integration and perception of pain.
  • the neural structure of the receptor; the limbic system with the highest density of receptors and the blue nucleus are mostly brain areas related to emotional and mental activities. They exert different effects by combining with opioid peptides in the brain or / and exogenous opioid alkaloids (morphine, naloxone, etc.).
  • Opioid peptide receptors can analgesic when stimulated, cause smooth muscle contraction, excite vascular motor centers and respiratory centers, and produce corresponding psychiatric symptoms.
  • the polypeptide of the present invention and the antagonist, agonist and inhibitor of the polypeptide can be directly used in the diagnosis and treatment of some diseases, including but not limited to:
  • Analgesia sedation, antitussive, cardiogenic asthma, diarrhea, constipation, gastrointestinal / urinary angina, mental illness (irritability, hallucinations, anxiety), acute poisoning of morphine analgesics (respiratory depression), etc. ;
  • Other disorders related to receptor dysfunction include:
  • Giant disease dwarfism, acromegaly (inhibitor of growth hormone release), hypercortisolism (Cushing's syndrome), primary hyperaldosteronism, secondary chronic adrenal insufficiency (promoting Adrenocortical hormone receptor), hyperthyroidism, hypothyroidism (thyrotropin receptor), male / female infertility, menstrual disorders (functional uterine bleeding, amenorrhea, polycystic ovary syndrome, premenstrual tension Syndrome, menopause syndrome), sexual development disorder (luteinizing hormone receptor, follicle stimulating hormone receptor), diabetes, hypoglycemia (glucagon receptor, insulin receptor), peptic ulcer, chronic digestion Bad (incretin receptors), etc .;
  • G protein has a very important role in the body, and its abnormal expression will cause abnormal proliferation of tissue cells and abnormal expression of protein.
  • the human G protein subunit is a component of G protein. Abnormal expression in the body can affect the formation of G protein, cause G protein dysfunction, cause abnormal proliferation of tissue cells and abnormal expression of proteins, and then cause the occurrence of related diseases.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human G protein subunit protein, and both have similar biological functions.
  • the polypeptide of the present invention is a component of the G protein in the body, and abnormal expression in the body can affect the formation of the G protein, cause dysfunction of the G protein, cause abnormal proliferation of tissue cells and abnormal expression of the protein, and then cause various tumor diseases.
  • the occurrence of these diseases includes but is not limited to: Common tumors of various tissues:
  • Papilloma squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (carcinoma) [breast, thyroid], mucinous / serous cystadenomas (carcinoma) [ovary], basal cell carcinoma [head and face Skin], (evil Sex) polytype adenoma [extending gland], papilloma, transitional epithelial cancer [bladder, renal pelvis], etc .; 2.
  • Mesenchymal tissue :
  • Malignant lymphoma [Neck, mediastinum, mesenteric and retroperitoneal lymph nodes], various leukemias [lymphoid hematopoietic tissue], multiple myeloma [push / thoracic / rib / skull and long bone], etc .;
  • Nerve fiber [systemic cutaneous nerve / deep nerve and internal organs], (malignant) schwannoma [nervous of head, neck, limbs, etc.], (malignant) glioblastoma [brain], medulloblastoma [ Cerebellum], (malignant) meningiomas [meninges], ganglioblastoma / neuroblastoma [mediastinum and retroperitoneum / adrenal medulla], etc .;
  • malignant melanoma skin, mucous membrane
  • (malignant) hydatidiform mole chorionic epithelial cancer [uterine]
  • (malignant) supporter cells stromal cell tumor
  • (malignant) granulosa cell tumor ovarian, testicular] fine Blastoma [testis], asexual cell tumor [ovary], embryonal cancer [testis, ovary], (malignant) teratoma [ovary, testis, mediastinum and palate tail], etc .
  • malignant melanoma skin, mucous membrane
  • hydatidiform mole chorionic epithelial cancer [uterine]
  • (malignant) supporter cells stromal cell tumor
  • (malignant) granulosa cell tumor ovarian, testicular] fine Blastoma [testis]
  • asexual cell tumor ovary
  • embryonal cancer testis, ovary
  • (malignant) teratoma
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of various diseases, such as various diseases related to hormone receptor instability, various tumors, and used for treating analgesia , Sedation, antitussive, cardiogenic asthma, diarrhea, constipation, gastrointestinal / urinary tract colic, mental illness (irritability, hallucinations, anxiety), acute poisoning of morphine analgesics (respiratory depression), etc.
  • various diseases such as various diseases related to hormone receptor instability, various tumors, and used for treating analgesia , Sedation, antitussive, cardiogenic asthma, diarrhea, constipation, gastrointestinal / urinary tract colic, mental illness (irritability, hallucinations, anxiety), acute poisoning of morphine analgesics (respiratory depression), etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) the human G protein subunit 9.46.
  • Agonists increase the human G protein subunit 9.46 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human G protein subunit 9.46 can be cultured with labeled human G protein subunit 9.46 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human G protein subunit 9. 46 include screened antibodies, compounds, receptor deletions and classes Like things. Antagonists of human G protein subunit 9. 46 can bind to human G protein subunit 9.46 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide such that the polypeptide cannot function biological functions.
  • human G protein subunit 9.46 When screening compounds as antagonists, human G protein subunit 9.46 can be added to the bioanalytical assay, and the compound can be determined by measuring the effect of the compound on the interaction between human G protein subunit 9.46 and its receptor Whether it is an antagonist. 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 the human G protein subunit 9.46 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 9.46 molecules of the human G protein subunit should be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human G protein subunit 9.46 epitope. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments from Fab expression libraries.
  • Polyclonal antibodies can be produced by direct injection of human G protein subunit 9.46 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 Adjuvant, etc.
  • Techniques for preparing monoclonal antibodies to human G protein subunit 9.46 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta- Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morris on 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 the human G protein subunit 9.46.
  • Antibodies against human G protein subunit 9. 46 can be used in immunohistochemical techniques to detect human G protein subunit 9. 46 in biopsy specimens.
  • Monoclonal antibodies that bind to the human G protein subunit 9.46 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.
  • the human G protein subunit 9.46 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 the antibody with a thiol cross-linking agent such as SPDP, and toxin is bound to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill the human G protein subunit 9.46 positive Cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to the human G protein subunit 9.46. 46.
  • the proper dose of antibody can stimulate or block the production or activity of human G protein subunit 9.46.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human G protein subunit 9.46.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human G protein subunit 9.46 detected in the test can be used to explain the importance of human G protein subunit 9.46 in various diseases and to diagnose the role of human G protein subunit 9.46. disease. '
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding the human G protein subunit 9.46 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 the human G protein subunit 9.46. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human G protein subunit 9.46 to inhibit endogenous human G protein subunit 9.46 activity.
  • a variant human G protein subunit 9.46 may be a shortened human G protein subunit 9.46 that lacks a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human G protein subunit 9.46.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding the human G protein subunit 9.46 into cells.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding the human G protein subunit 9.46 can be found in the literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding the human G protein subunit 9.46 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 the human G protein subunit 9.46 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 of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond rather than the phosphate Ester bond. '
  • the polynucleotide encoding the human G protein subunit 9. 46 can be used for the diagnosis of diseases related to the human G protein subunit 9. 46.
  • a polynucleotide encoding the human G protein subunit 9. 46 can be used to detect the expression of the human G protein subunit 9. 46 or the abnormal expression of the human G protein subunit 9. 46 in a disease state.
  • the DNA sequence encoding the human G protein subunit 9. 46 can be used to hybridize biopsy specimens to determine the expression of the human G protein subunit 9. 46.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microar ray) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis of genes in tissues and genes. diagnosis.
  • Human G protein subunit 9.46 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human G protein subunit 9, 46 transcripts.
  • Human G protein subunit 9.46 gene can also be used to diagnose human G protein subunit 9.46-related diseases.
  • Human G protein subunit 9.46 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human G protein subunit 9. 46 DNA sequences. Mutations can be detected using existing techniques such as Sou thern blotting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the use of Nort Hern blotting and Western blotting can indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • a PCR primer (preferably 15-35b P ) is prepared based on the cDNA, and the sequence can be mapped on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells 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 in a similar manner, 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 of cDNA clones and metaphase chromosomes allows precise chromosomal localization in one step.
  • 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 in, for example, V. Mckusick, Mendel ian 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 are 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 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 chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • the human G protein subunit 9.46 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of 9.46 human G protein subunits to be 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.

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Abstract

L'invention concerne un nouveau polypeptide, une sous-unité humaine 9.46 de protéine G, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment de maladies liées aux dysfonctionnements des récepteurs hormonaux, de toutes sortes de tumeurs et comme analgésique, sédatif, antitussif dans le traitement de l'asthme, de diarrhées, de la constipation, de coliques du tractus gastro-intestinal et des voies urinaires, de maladies psychologiques (agitation, hallucinations et anxiété) et de l'intoxication due à l'ingestion d'analgésiques de type morphine (problèmes respiratoires). L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la sous-unité humaine 9.46 de protéine G.
PCT/CN2001/000985 2000-06-19 2001-06-18 Nouveau polypeptide, sous-unite humaine 9.46 de proteine g, et polynucleotide codant ce polypeptide Ceased WO2002004504A1 (fr)

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AU93622/01A AU9362201A (en) 2000-06-19 2001-06-18 A novel polypeptide - human g-protein subunit 9.46 and a polynucleotide encodingthe same

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CN 00116603 CN1329061A (zh) 2000-06-19 2000-06-19 一种新的多肽——人g蛋白亚基9.46和编码这种多肽的多核苷酸
CN00116603.4 2000-06-19

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011023390A1 (fr) * 2009-08-28 2011-03-03 F. Hoffmann-La Roche Ag Anticorps contre cdcp1 destinés au traitement du cancer
US8394928B2 (en) 2009-08-28 2013-03-12 Roche Glycart Ag Humanized anti-CDCP1 antibodies

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998025956A2 (fr) * 1996-12-12 1998-06-18 Incyte Pharmaceuticals, Inc. Nouvelles proteines humaines se liant a la guanosine triphosphate
US5871971A (en) * 1997-07-17 1999-02-16 Incyte Pharmaceuticals, Inc. Human developmentally regulated GTP-binding protein

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998025956A2 (fr) * 1996-12-12 1998-06-18 Incyte Pharmaceuticals, Inc. Nouvelles proteines humaines se liant a la guanosine triphosphate
US5871971A (en) * 1997-07-17 1999-02-16 Incyte Pharmaceuticals, Inc. Human developmentally regulated GTP-binding protein

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011023390A1 (fr) * 2009-08-28 2011-03-03 F. Hoffmann-La Roche Ag Anticorps contre cdcp1 destinés au traitement du cancer
US8394928B2 (en) 2009-08-28 2013-03-12 Roche Glycart Ag Humanized anti-CDCP1 antibodies
US8883159B2 (en) 2009-08-28 2014-11-11 Hoffmann-La Roche, Inc. Antibodies against CDCP1 for the treatment of cancer
US9346886B2 (en) 2009-08-28 2016-05-24 Roche Glycart Ag Humanized anti-CDCP1 antibodies

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AU9362201A (en) 2002-01-21

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