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WO2002000712A1 - Nouveau polypeptide, recepteur cannabinoide central humain 22, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, recepteur cannabinoide central humain 22, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002000712A1
WO2002000712A1 PCT/CN2001/000758 CN0100758W WO0200712A1 WO 2002000712 A1 WO2002000712 A1 WO 2002000712A1 CN 0100758 W CN0100758 W CN 0100758W WO 0200712 A1 WO0200712 A1 WO 0200712A1
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Prior art keywords
polypeptide
polynucleotide
human central
component receptor
sequence
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PCT/CN2001/000758
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Application filed by Shanghai Biowindow Gene Development Inc. filed Critical Shanghai Biowindow Gene Development Inc.
Priority to AU87499/01A priority Critical patent/AU8749901A/en
Publication of WO2002000712A1 publication Critical patent/WO2002000712A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, the human central cannabis component receptor 22, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background
  • Cannabis has a wide range of effects on the central nervous system.
  • the pharmacological activity of cannabis, its most active ingredient, ⁇ -tetrahydrocannabinol, the endogenous astringent anandamide, and many artificial astringents are mediated by the cannabino id receptor.
  • the cannabis component receptor and its endogenous ligands form a system that regulates certain brain functions, such as brain injury, motion control, memory, and neuroendocrine control. Recent discoveries suggest that this system also plays a role in brain development [Trends Neurosci. 2000 Jan; 23 (1): 14-20]. Most motor dysfunction and motor dysfunction are caused by dysfunction of the basal ganglia-thalamus-cortex pathway.
  • the central cannabis component receptor system may play a role in the occurrence of these diseases.
  • cannabis is used in the treatment of Touret te syndrome, a psychosis that shows ataxia, speech disorders and convulsions. Parkinson's disease, tremors and strain due to reduced dopamine Curative effect in disorders [Forsch Komplementarraed 1999 Oct; 6 Suppl 3: 23_7].
  • CDM for human cannabis receptors has been isolated.
  • the protein inferred from this has 472 amino acids, and its seven highly hydrophobic parts are typical features of the G protein coupled receptor superfamily, indicating that the hemp component receptor is a G protein.
  • the central cannabis component receptor CBl (centra l cannabinoid receptor) mediates the inhibition of adenylate cyclase and the inhibition of N-type calcium ion channels, and also mediates the activation of extracellular signal-regulated kinases.
  • CB1 activation inhibits the release of synaptic transmitters in the rat hippocampus [Brain Res 2000 Jan 10; 852 (2): 398-405].
  • CBl and its mRNA are mainly found in the brain, they are also found in peripheral tissues, such as in the testis, spleen, and white blood cells [Eur. J. Biochem. 214, 173-180].
  • the central cannabis component receptor CB2 is mainly expressed in the immune system, and it has also been reported to be found in the mouse cerebellum.
  • the sequence of the transmembrane region of CB2 is only 51% similar to CB1, but its properties of binding to different ligands are very similar to CB1.
  • CB2 like CB1, can also inhibit adenylate cyclase [FEBS Let t. 350, 240-244].
  • CBl and CB2 are in the retina They were all found [Vis Neurosc i 2000 Jan- Feb; 17 (1): 91-5].
  • CB1 levels are lower at birth. After birth, at least in certain regions of the brain, CB1 levels rise until reaching adult levels. After that, the level of CB1 dropped again. This decrease may be caused by the lower translation rate of CB1 or the instability of mRM [Neurosc i. Let t. 147, 179-181].
  • the human central cannabis component receptor 22 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. Therefore, there is always a need to identify more involved in these During the process of the human central hemp component receptor 22 protein, particularly the amino acid sequence of this protein was identified.
  • the isolation of the new central cannabis component receptor 22 protein encoding gene 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 code for DM.
  • 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 the human central cannabis component receptor 22.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human central cannabis component receptor 22.
  • Another object of the present invention is to provide a method for producing the human central cannabis component receptor 22.
  • Another object of the present invention is to provide an antibody against the human central cannabis component receptor 22 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the human central cannabis component receptor 22 of the polypeptide of the present invention.
  • Another object of the present invention is to provide diagnostic treatment related to abnormality of human central cannabis component receptor 22 Of disease.
  • 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 156 to 761 in SEQ ID NO: 1; and (b) having a sequence in SEQ ID NO: .1 1- 2802-bit sequence.
  • 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 central cannabis component receptor 22 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to the abnormal expression of the human central cannabis component receptor 22 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting 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 preparation of the polypeptide and / or polynucleotide of the present invention for the treatment of neuropsychiatric disorders, developmental disorders, tumors or occupying lesions of the nervous system, immune diseases, inflammation, HIV infection, etc., especially It is used for treating mental disorders caused by psychoactive drugs or other diseases caused by abnormal expression of human central cannabis component receptor 22.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the central cannabis component receptor 22 and the central cannabis component receptor of the present inventors.
  • the upper graph is a graph of the expression profile of the central cannabis component receptor 22, and the lower graph is the graph of the expression profile of the central cannabis component receptor.
  • 1 indicates fetal kidney
  • 2 indicates fetal large intestine
  • 3 indicates fetal small intestine
  • 4 indicates fetal muscle
  • 5 indicates fetal brain
  • 6 indicates fetal bladder
  • 7 indicates unstarved L02
  • 8 indicates L02 +, lhr, As 3+
  • 9 indicates ECV304 PMA-
  • 10 means ECV304 PMA +
  • 11 means fetal liver
  • 12 means normal liver
  • 13 means thyroid
  • 14 means skin
  • 15 means fetal lung
  • 16 means lung
  • 17 means lung cancer
  • 18 means fetal spleen
  • 19 means spleen
  • 20 Indicates prostate
  • 21 indicates fetal heart
  • 22 indicates heart
  • 23 indicates muscle
  • 24 indicates testis
  • 25 indicates fetal thymus
  • 26 indicates thymus.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human central hemp component receptor 22.
  • 22KDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • 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” or “addition” 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.
  • Biological activity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • the term “immunologically active” refers to the ability of natural, recombinant, or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when bound to the human central cannabis component receptor 22, causes a change in the protein to regulate the activity of the protein.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to the human central cannabis component receptor 22.
  • Antagonist refers to a molecule that can block or modulate the biological or immunological activity of the human central cannabis component receptor 22 when bound to the human central cannabis component receptor 22.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to the human central cannabis component receptor 22.
  • Regular refers to a change in the function of the human central cannabis component receptor 22, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of the human central cannabis component receptor 22. change.
  • substantially pure means 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 central cannabis component receptor 22 using standard protein purification techniques.
  • the substantially pure human central hemp component receptor 22 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human central hemp component receptor 22 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • 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 Northern 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 the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • 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 (Hi ggins, DG and PM Sharp (1988) Gene 73: 237-244) 0 Clus ter method by examining the distances between all pairs of each set of sequence 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 percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in enzymology 183: 625-645).
  • 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 substitution for example, negatively charged amino acids 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 HFP or a chemical modification of its nucleic acid. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and?, which can specifically bind to the epitope of human central hemp component receptor 22.
  • 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 occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, 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 a component 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 existing in the natural state.
  • isolated human central cannabis component receptor 22 means that human central cannabis component receptor 22 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated.
  • Those skilled in the art can purify the human central hemp component receptor 22 using standard protein purification techniques.
  • Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human central hemp component receptor 22 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide ⁇ central hemp component receptor 22, 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 produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of the human central cannabis component receptor 22.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human central cannabis component receptor 22 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ )
  • Such a polypeptide sequence 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
  • a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as the leader or secretory sequence or the sequence used to purify the polypeptide or protease sequence).
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 2802 bases, and its open reading frame 156-761 encodes 201 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile to the central cannabis component receptor, and it can be deduced that the human central cannabis component receptor 22 has a similar function as the central cannabis component receptor.
  • the polynucleotide of the present invention may be in the form of DM or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region shown in SEQ ID NO: 1
  • the sequences are identical or degenerate variants.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% ( ⁇ / ⁇ ) formamide, 0.1% calf serum / 0.1% Fi col l, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, 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 human central cannabis component receptor 22.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human central cannabis component receptor 22 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DM fragment sequence of the present invention can also be obtained by the following methods: 1) Isolation of double-stranded DNA from genomic DM Sequence; 2) chemically synthesize a DM sequence to obtain a double-stranded DM of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDM of interest is to isolate mRM from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua 1, Cold Spruing Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybrids; (2) the presence or absence of marker gene functions; (3) determining the level of the transcript of the human central cannabis component receptor 22; ( 4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (BLISA) can be used to detect protein products expressed by the human central cannabis component receptor 22 gene expression protein.
  • BLISA enzyme-linked immunosorbent assay
  • a method (Sa iki, et al. Science 1985; 230: 1350-1354) using PCR technology to amplify DM / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to 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 vector or a direct use of the vector of the present invention.
  • a polynucleotide sequence encoding the human central cannabis component receptor 22 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding the human central cannabis component receptor 22 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombination DM technology, DM synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spin Harbor Laboratory. New York, 1989).
  • the MA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • the expression vector preferably incorporates one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and Green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and Green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding the human central cannabis component receptor 22 or a polynucleotide containing the same can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DM can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a 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.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human central hemp component receptor 22 (Science, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Cannabis has a wide range of effects on the central nervous system. Cannabis component receptor and its endogenous ligand set It has become a system that regulates certain brain functions, such as brain injury, motion control, memory and neuroendocrine control, etc. Recently, it has been found that this system also plays a role in brain development.
  • the central cannabis component receptor CB1 mediates the inhibitory effect of adenylate cyclase and the N-type calcium ion channel, and also mediates the activation of extracellular signal-regulated kinases. CB1 activation inhibits the release of synaptic transmitters in the rat hippocampus. CB1 is mainly found in the brain, but is also found in peripheral tissues such as testes, spleen, and white blood cells. Most motor dysfunction and motor dysfunction are caused by dysfunction of the basal ganglia-thalamus-cortex pathway. Because the endocannabinoid component plays a role in controlling exercise, the central cannabis component receptor system may play a role in the occurrence of these diseases. There is now evidence that cannabis is effective in the treatment of Tourette's disease, Parkinson's disease, Alzheimer's disease, obsessive-compulsive behavior disorder, tremor and dystonia.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human central hemp component receptor CB1, both of which have similar biological functions. It mainly mediates the pharmacological activity of cannabis in the body, especially in the nervous central tissues, which is important for the regulation of neurotransmitters, and is also important for the regulation of the body's immune system and for the regulation of growth and development. Its abnormal expression is closely related to the occurrence of pathological processes of the above-mentioned tissue systems, and causes related diseases.
  • the abnormal expression of the human central cannabis component receptor 22 of the present invention will produce various diseases, especially neuropsychiatric disorders, immune diseases, development disorders, inflammation, and various tumors. These diseases include but are not limited to :
  • -Neuropsychiatric disorders Tourette's disease, Alzheimer's disease, Parkinson's disease, obsessive-compulsive disorder, chorea, depression, amnesia, Huntington's disease, epilepsy, migraine, multiple sclerosis , Schizophrenia, depression, neurasthenia, neuromuscular disease, neurocutaneous syndrome, trigeminal neuralgia, facial paralysis
  • Developmental disorders neural tube insufficiency, brain developmental abnormalities, neuronal migration disorders, congenital abortion, cleft palate, limb absentness, limb differentiation disorders, atrial septal defect, neural tube defects, congenital hydrocephalus, mental retardation, Brain development disorders, skin, fat and muscular dysplasia, bone and joint dysplasia, sexual retardation
  • Tumors or occupying lesions of the nervous system Tumors or occupying lesions of the nervous system: neuroblastoma, astrocytoma, ependymoma, glioblastoma, neurofibromas, intracranial granulomas ⁇
  • Immune diseases Guillain-Barre syndrome, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, myasthenia gravis, common variable immunodeficiency disease, primary B lymphocyte immunodeficiency disease, acquired immunity Defect Syndrome
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebral spinal cord Multiple sclerosis, Glomerulonephritis, Myocarditis, Cardiomyopathy, Atherosclerosis, Gastric ulcer, Cervicitis, Some infectious inflammations
  • Abnormal expression of the human central cannabis component receptor 22 of the present invention may also cause certain genetic diseases and the like.
  • the polypeptide of the present invention, as well as the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially neuropsychiatric disorders, tumors or occupying lesions of the nervous system, and development disorders Disease, immune disease, inflammation, certain hereditary diseases, etc.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used for the treatment of mental disorders caused by psychoactive drugs.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) the human central hemp component receptor 22.
  • Agonists enhance human central cannabis component receptor 22 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing the human central cannabis component receptor 22 can be cultured with the labeled human central cannabis component receptor 22 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human central cannabis component receptor 22 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of the human central cannabis component receptor 22 can bind to the human central cannabis component receptor 22 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot exert its biology Features.
  • human central cannabis component receptor 22 can be added to a bioanalytical assay to determine whether a compound is a compound by measuring its effect on the interaction between human central cannabis component receptor 22 and its receptor. Antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to the human central cannabis component receptor 22 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, the human central cannabis component receptor 22 molecule should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human central cannabis component receptor 22 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by direct injection of human central cannabis component receptor 22 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 Agent.
  • Techniques for preparing monoclonal antibodies to human central hemp component receptor 22 include, but are not limited to Based on hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Antibodies to human central cannabis component receptor 22 can be used in immunohistochemical techniques to detect human central cannabis component receptor 22 in biopsy specimens.
  • Monoclonal antibodies that bind to the human central cannabis component receptor 22 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human central cannabis component receptor 22 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 sulfhydryl crosslinker such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human central cannabis component receptor 22 positive cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to the human central cannabis component receptor 22.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of the human central cannabis component receptor 22.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human central cannabis component receptor 22 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The levels of human central cannabis component receptor 22 detected in the test can be used to explain the importance of human central cannabis component receptor 22 in various diseases and to diagnose diseases where human central cannabis component receptor 22 functions.
  • 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.
  • Polynucleotides encoding the human central cannabis component receptor 22 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of human central cannabis component receptor 22.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human central cannabis component receptor 22 to inhibit endogenous human central cannabis component receptor 22 activity.
  • a variant human central cannabis component receptor 22 may be a shortened human central cannabis component receptor 22 lacking a signaling functional domain. Although it can bind to a downstream substrate, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of the human central cannabis component receptor 22.
  • Virus-derived expression vectors such as retroviruses, adenoviruses, adenopathies Poison-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding the human central cannabis component receptor 22 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding the human central cannabis component receptor 22 can be found in the existing literature (Sambrook, eta l.).
  • a recombinant polynucleotide encoding human central cannabis component receptor 22 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 RM and DNA
  • ribozymes that inhibit human central cannabis component receptor 22 raRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target MA to perform endonucleation.
  • Antisense RM and D and ribozymes can be obtained by any existing RNA or DM synthesis technology. For example, the technology of solid phase phosphoric acid amide synthesis of oligonucleotides has been widely used.
  • Antisense RM molecules can be obtained by in vitro or in vivo transcription of the D sequence encoding the RNA.
  • This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human central cannabis component receptor 22 can be used for the diagnosis of diseases related to human central cannabis component receptor 22.
  • the polynucleotide encoding the human central cannabis component receptor 22 can be used to detect the expression of the human central cannabis component receptor 22 or the abnormal expression of the human central cannabis component receptor 22 in a disease state.
  • the DNA sequence encoding human central cannabis component receptor 22 can be used to hybridize biopsy specimens to determine the expression of human central cannabis component receptor 22.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and the relevant kits are commercially available.
  • RNA-polymerase chain reaction (RT-PCR) in vitro amplification using human central cannabis component receptor 22-specific primers can also detect human central cannabis component receptor 1 transcription products.
  • Detection of mutations in the human central cannabis component receptor 22 gene can also be used to diagnose human central cannabis component receptor 22-related diseases.
  • Human central cannabis component receptor 22 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human central cannabis component receptor 22 DNA sequence. Mutations can be detected using well-known techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Nor thern blotting, Wes tern Blotting can indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those 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 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 with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the difference in cDM 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).
  • 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 contains a safe and effective amount of the polypeptide or antagonist and does not affect Pharmaceutically effective carriers and excipients. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human central cannabis component receptor 22 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human central cannabis component receptor 22 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. Examples
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRM was isolated from total RNA using Quik mRNA Isolat ion Kit (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 00 fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5a.
  • the bacteria formed a CDM library.
  • Dye terminate cycle reaction ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined CDM sequence was compared with the existing public D sequence database (Genebank), and it was found that the CDM sequence of one of the clones 0373a07 was new DNA.
  • a series of primers were synthesized to perform bidirectional determination of the inserted CDM fragments contained in this clone.
  • the 0373a07 clone contained a full-length cDNA of 2802bp (as shown by Seq ID NO: l), and a 606bp open reading frame (0RF) from 156bp to 761bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-0373a07 and the encoded protein was named human central hemp component receptor 22.
  • Example 2 Cloning of a gene encoding human central hemp component receptor 22 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Primer 1 5,-AACCTGCCTGACACCCTCACGCTC -3, (SEQ ID NO: 3)
  • Primer2 5'- AACCTGCCTGACACCCTCACGCTC -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • a reaction volume of 50 ⁇ l contains 50 mmol / L: Cl, l (tonol / L Tri s-HCl pH8.5, 1.5 mol / L MgCl 2 , 200 ⁇ 1/1 dNTP, lOpmol primers, 1U of Taq DM polymerase (product of Clontech).
  • the reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94.C 30sec; 55.C 30sec; 72 ° C 2min.
  • ⁇ -act in was used as a positive control and template blank was used as a negative control.
  • Amplification products were purified using a QIAGEN kit and TA cloning kits were connected to a pCR vector (Invitrogen).
  • DM sequence analysis The results show that the DM sequence of the PCR product is exactly the same as 1- 2802 bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human central hemp component receptor 22 gene expression The total RM was extracted in one step [Anal. Biochem 1987 162, 156-159]. This method includes acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate and 0.2M sodium acetate (pH4.0).
  • a 2 2 P dATP was used to prepare a 32 P-labeled DNA probe by random primers.
  • the DNA probe used was the PCR-encoded human central hemp component receptor 22 coding region sequence (156bp) shown in FIG. 1 To 761bp).
  • 32P-labeled probes (approximately 2 x 10 6 cpm / ml) were hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7. 4) -5 SSC-5 ⁇ Denhardt's solution and 20 ⁇ g / ml salmon sperm DNA.
  • the PCR reaction was performed using the pBS-0373a07 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0373a07 plasmid, primers Primer-3 and Primer-4 were 1 Opmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles.
  • Ndel and 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 the colibacillus DH5c by the calcium chloride method. After being cultured overnight in LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0373a07) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • a peptide specific to the human central hemp component receptor 22 was synthesized using a peptide synthesizer (product of PE): NH2-Met-I le-Phe-Ser-Cys-Phe-Cys-Lys-Ala-His-Pro-I le-Pro-Pro-Leu-C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • the suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects.
  • the probes can be used to hybridize to the genome or CDM library 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 tissues or Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by using a filter hybridization method.
  • 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), so that the hybridization background is reduced and only strong specific signals are retained.
  • 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 from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • 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 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (bond) 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 membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the 32 PP r0 be (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • the sample membrane was placed in a plastic bag, and 3 to 10 mg of prehybridization solution (lOxDenhardt-s; 6xSSC, 0.1 lrag / ml CT DM (calf thymus DM)) was added. After sealing the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution lOxDenhardt-s; 6xSSC, 0.1 lrag / ml CT DM (calf thymus DM)
  • 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 chip or gene microarray is a new technology 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 a target DM for gene chip technology for high-throughput research of new gene functions; searching for and screening 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.
  • 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 respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500 ng / ul after purification, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ . The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified with Oligotex mRNA Midi Ki t (purchased from QiaGen).
  • Cy3dUTP (5-Amino-propargyl— 2'-deoxyuridine 5> -triphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5- Amino- propargyl-2 ' -deoxyuridine 5'-triphate coupled to Cy5 f luorescent dye, purchased from Amershara Phamacia Biotech The company) labeled the mRNA of specific tissues (or stimulated cell lines) of the body and prepared probes after purification.
  • fluorescent reagent Cy5dUTP (5- Amino- propargyl-2 ' -deoxyuridine 5'-triphate coupled to Cy5
  • the probes from the two types of tissues were hybridized with the chip in a UniHyb TM Hybridization Solution (purchased from TeleChera) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was then performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed by Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, and non-starved L02 cell line , L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain Fetal lung and fetal heart.

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Abstract

L'invention concerne un nouveau polypeptide, un récepteur cannabinoïde central humain 22, 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 des maladies liées à des troubles neuropsychologiques, des troubles du développement, des tumeurs du système nerveux ou des lésions occupant de l'espace, des maladies immunitaires, des inflammations, de l'infection par VIH et plus particulièrement des maladies psychologiques provoquées par l'ingestion de médicaments destinés à traiter les troubles psychologiques. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant le récepteur cannabinoïde central humain 22.
PCT/CN2001/000758 2000-05-16 2001-05-14 Nouveau polypeptide, recepteur cannabinoide central humain 22, et polynucleotide codant ce polypeptide WO2002000712A1 (fr)

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CN 00115715 CN1323822A (zh) 2000-05-16 2000-05-16 一种新的多肽——人中枢大麻成分受体22和编码这种多肽的多核苷酸
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992002640A1 (fr) * 1990-08-08 1992-02-20 The United States Of America, Represented By The Secretary, United States Department Of Commerce Recepteur de cannabinoides
WO1994025589A1 (fr) * 1993-04-27 1994-11-10 Medical Research Council Recepteur des cannabinoïdes exprime dans les cellules du systeme immunitaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992002640A1 (fr) * 1990-08-08 1992-02-20 The United States Of America, Represented By The Secretary, United States Department Of Commerce Recepteur de cannabinoides
WO1994025589A1 (fr) * 1993-04-27 1994-11-10 Medical Research Council Recepteur des cannabinoïdes exprime dans les cellules du systeme immunitaire

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BOUABOULA M. ET AL., BIOCHEM. J., vol. 312, no. PT. 2, 1 December 1995 (1995-12-01), pages 637 - 641 *
DATABASE GENBANK [online] 14 March 2000 (2000-03-14), RUIZ-PEREZ V.L. ET AL., Database accession no. AF216185 *
DATABASE GENBANK [online] 21 March 2000 (2000-03-21), GALDZICKA M. ET AL., Database accession no. AF239742 *

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