WO2005033308A1 - Proteine associee a la schizophrenie et gene codant pour cette derniere - Google Patents

Proteine associee a la schizophrenie et gene codant pour cette derniere Download PDF

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Publication number
WO2005033308A1
WO2005033308A1 PCT/JP2004/006340 JP2004006340W WO2005033308A1 WO 2005033308 A1 WO2005033308 A1 WO 2005033308A1 JP 2004006340 W JP2004006340 W JP 2004006340W WO 2005033308 A1 WO2005033308 A1 WO 2005033308A1
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protein
schizophrenia
expression level
seq
antibody
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PCT/JP2004/006340
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English (en)
Japanese (ja)
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Toru Nishikawa
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Toru Nishikawa
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a schizophrenia-related protein, a gene encoding the protein, a recombinant vector containing the gene, a protein transformant containing the recombinant vector, an antibody against the protein or a fragment thereof, and a gene encoding the gene.
  • a Method for diagnosing schizophrenia or schizophrenia-like abnormalities using endogenous expression level as an indicator 3 ⁇ 4 and diagnostic kit, and schizophrenia or schizophrenia-like with the effect of reducing the expression level of the above genes in the brain The present invention relates to a method for screening a therapeutic substance and a screening kit.
  • neural circuit liquors neural information processing systems
  • Drugs that cause schizophrenia-like symptoms include stimulants such as amphetamine and methamphetamine; dopamine agonists such as cocaine; NMDA-type glutamate receptors such as phencyclidine and ketamine A body blocker is known (Toru Nishikawa, Psychiatry Review, Separate Volume, “Considering Schizophrenia for the 21st Century” (Gusyuo * Kenichi Omori), 1994, p. 26-37). It is likely that these drugs cause symptoms very similar to schizophrenia, either directly or indirectly, by causing abnormalities in information processing systems impaired by schizophrenia.
  • schizophrenia-related genes can be identified by searching for genes that acquire a development-dependent response to schizophrenia-like symptoms (Toru Nishikawa, Psychiatric Neurology Journal) 2002, Vol. 104, No. 6, p. 487-492). Disclosure of the invention
  • the present invention firstly relates to a schizophrenia-related protein, It is an object of the present invention to provide a gene to be transferred, a recombinant vector containing the gene, a transformant containing the recombinant vector, and an antibody against the protein or a fragment thereof.
  • the present invention provides a method and a kit for diagnosing schizophrenia or schizophrenia-like abnormalities, using the expression level in the brain of a gene encoding a schizophrenia-related protein as an index.
  • the purpose is to: '
  • the present invention provides a method for screening and treating a substance for preventing and treating schizophrenia or a schizophrenia-like abnormality using the effect of reducing the expression level of a gene encoding a schizophrenia-related protein as an index.
  • the purpose is to provide a kit.
  • the present invention provides the following proteins, genes, recombinant vectors, transformants, antibodies or fragments thereof, diagnostic methods and kits, and screening methods and kits.
  • (b) consists of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 or 6, and the basic brain expression level decreases in a development-dependent manner Protein
  • the protein shown in (d) is a protein whose expression level in the brain is increased in a development-dependent manner by administration of a drug that causes a schizophrenia-like abnormality.
  • a kit for diagnosing schizophrenia or schizophrenia-like abnormality comprising an oligonucleotide or polynucleotide capable of hybridizing to the nucleic acid encoding the protein shown in (c) or (d).
  • a kit for diagnosing schizophrenia or schizophrenia-like abnormality comprising an antibody or a fragment thereof capable of reacting with the protein shown in the above (c) or (d).
  • Schizophrenia or schizophrenia using as an index the effect of reducing the expression level of the gene encoding the protein shown in (c) or (d) above in a cell or tissue in which the expression level of the gene is elevated.
  • Prevention of candidate substances against dysplasia ⁇ A method for screening schizophrenia or schizophrenia-like abnormalities, including the step of determining the therapeutic effect.
  • Kit for screening of schizophrenia or schizophrenia-like abnormalities including screening body for schizophrenia or schizophrenia-like abnormalities, including a bovine or a fragment thereof capable of reacting to the protein shown in (c) or (d) above.
  • FIG. 1A shows the results of detecting mRNA from neocortex of mature rats (50 days old) and young rats (8 days old) treated with saline or MAP by RNA fingerprinting using arbitrarily primed PCR.
  • FIG. 1B is a schematic diagram of the structure of rat mrtl cDNA
  • FIG. 1C is a northern blot of poly (A) + RNA (2 ⁇ g / lane) derived from adult rat neocortex.
  • FIG. 1D shows the results from various brain sites and peripheral organs using cDNA probes for IVS, B41-3 or 0-actin.
  • FIG. 9 is a view showing the results of Northern plot of (A) + RNA (1 g / lane).
  • FIG. 2A is a schematic diagram showing the structure of Mrtla and MrUb and the recognition site of each antibody
  • FIG. 2B is extracted from neocortex of a young rat (8 days old) and a mature rat (50 days old).
  • FIG. 2C shows the Western blot results of 25 protein samples, and FIG. 2C shows the amino acid sequences at the 'C-terminus of MrUa and MrUb corresponding to the peptides used for preparing the isoform-specific anti-Mrtl antibody.
  • FIG. 2D shows the results of the characterization of Mrtlb-specific antibodies, and FIG. 2E shows the subcellular localization of Mrtl proteins in neocortical tissue. .
  • Figure 3A is a schematic diagram of the recognition site of the mrtl mutant-specific primer and the putative PCR product.
  • Figure 3B shows the PCR products amplified by the specific primer set for mrtla and mrtlb in various samples.
  • FIG.3C shows the results of co-amplification RT-PCR of rat mrtlb-specific sequence and 28S liposomal RNA (rRNA) -specific sequence as an internal control
  • FIG.3D shows FIG. 4 is a graph showing mrtl a mRNA expression levels and mrtlb mRNA expression levels one hour after acute MAP administration (4.8 mg / kg, sc).
  • FIG.4A is a diagram showing the expression levels of mrtlb mRNA 1 hour, 3 hours, 6 hours, and 24 hours after a single MAP administration
  • FIG.4B is the total mRNA expression from 8 to 50 days after birth
  • FIG. 4C and FIG. 4D show the effects of acute administration of various drugs on mrtlb mRNA expression levels
  • FIGS. 5A and 5B are diagrams showing the effects of D1 antagonists on changes in neocortical basal mrtlb mRNA expression levels induced by repeated MAP administration.
  • the protein of the present invention is a protein shown in the following (a), (b), (c) or (d).
  • protein (a) a protein comprising the amino acid sequence of SEQ ID NO: 2 or 6 (hereinafter sometimes referred to as “protein (a)”)
  • Protein an amino acid sequence represented by SEQ ID NO: 2 or 6, wherein one or more amino acids are deleted, substituted or added, and the basic brain expression level decreases in a development-dependent manner Protein (hereinafter "protein
  • protein (c) a protein consisting of the amino acid sequence of SEQ ID NO: 4 or 8 (hereinafter sometimes referred to as “protein (c)”)
  • protein (d) one or more amino acids in the amino acid sequence of SEQ ID NO: 4 or 8 are deleted, substituted or added, and the basic brain expression level decreases in a development-dependent manner Protein (hereinafter sometimes referred to as “protein (d)”)
  • the protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4 is a protein derived from human, and the protein consisting of the amino acid sequence of SEQ ID NO: 6 or 8 is a protein derived from mouse.
  • Proteins having the amino acid sequence of SEQ ID NO: 2 or 6 contained in the protein (a) are of the same kind (referred to as “Mrtla” or “Mrtla protein” in Examples described later), and are included in the protein (c).
  • the protein comprising the amino acid sequence of SEQ ID NO: 4 or 8 is a protein of a different type from the protein (a) (referred to as “Mrtlb” or “Mrtlb protein” in Examples described later), and a protein ( a) and (c) relate to splicing variants.
  • basal brain expression levels decrease in a development-dependent manner.
  • “basic brain expression level” refers to drugs Expression level in the brain in a state that is not processed (natural state) by cerebral neocortex, cerebral neocortex, striatum, hippocampus, cerebellum, limbic forebrain, thalamus And expression levels in various parts or parts of the brain such as the hypothalamus, midbrain and brainstem.
  • development-dependent decrease means that the basic brain expression level decreases as the child develops from infancy to maturity. Basic expression levels in the brain after reaching the transition from childhood to maturity are almost constant.
  • the juvenile, transitional and maturation stages vary according to the species of the animal, but the juvenile period of rats is usually from 0 to about 21 days after birth, and the mature period of rats is usually after birth. Approximately 35 to about 42 days or more, human immature is usually 0 to about 6 years old, and human maturity is usually about 18 to about 20 years old.
  • the transition period of humans includes puberty, and puberty is usually about 10 to about 18 years after birth.
  • Both proteins (a) and (c) have a PDZ domain and a PX domain in order from the N-terminal side.
  • both the PDZ domain and the PX domain are functional domains that play a role in mediating protein-protein interaction, and each of the amino acid sequences of SEQ ID NOs: 2, 4, 6, or 8 has 45 to 13 36
  • the amino acid sequence at the 4th, 45th to 13th, 43rd to 134th or 43th to 134th positions corresponds to the PDZ domain, and the 15th to 26th at the 15th to 15th to 16th, respectively.
  • the amino acid sequence at position 265, 154 to 263 or 154 to 263 corresponds to the PX domain.
  • the PDZ domain consists of six) 3 sheets and two helices, often recognizing the short C-terminal sequence of the target molecule, and is called a class I binding motif, Ser / Thr-X-Val
  • the consensus sequence is / Leu / Ile (Songyang, ⁇ ⁇ et al., Science, 275: 73-76, 1997).
  • the PDZ domain also binds to the class II binding motif (Phe / Tyr-X-Phe / Val / Ala / IIe) found on the erythrocyte membrane protein glycophorin C, which is the target molecule of P55. (Songyang, Z. et al., Science, 275: 73-76, 1997).
  • the PDZ domain binds to the PDZ domain of another molecule, and the PDZ domain binds to another functional domain (for example, guanylate kinase domain) in the same molecule.
  • Functions of the PDZ domain include, for example, clustering of membrane proteins, molecular accumulation, participation of membrane proteins in transport and endocytosis, integration of signal transduction, crosstalk, amplification, and suppression.
  • the PX domain is found as a domain of approximately 140 amino acids conserved in the NADPH oxidase subunits p47hox and P40phox, and to date, over 170 eukaryotic proteins (e.g., phospholipase D, Nexin that regulates vesicle trafficking) CISK, which is involved in cell survival signals, Bps5, Vpsl7, Vam7 and Mvpl, which are involved in membrane transport of Sacc haromyces cerevesiae, and Beml which regulates yeast budding).
  • the PX domain of p47 ox consists of three
  • Protein (c) increases the expression level in the brain in a development-dependent manner by administration of a drug that causes schizophrenia-like abnormalities, while protein (a) increases the expression level in the brain by administering a drug that causes schizophrenia-like abnormalities. Also, the expression level in the brain does not increase in a development-dependent manner.
  • schizophrenia-like abnormalities refers to abnormalities similar to those exhibited by schizophrenia patients (humans) (abnormalities as a schizophrenia model). Examples of abnormalities exhibited by hallucinations, delusions, and psychomotor excitement include abnormalities such as hallucinations, delusions, and psychomotor excitement. Abnormalities.
  • “Drugs causing schizophrenia-like abnormalities” include, for example, stimulants such as amphetamine and methamphetamine, or W 200 derivatives; dopamine agonists such as central stimulants such as cocaine, methylphenidite or derivatives thereof; L-D0PA, promocribtin, apomorphine and the like.
  • “Administration” includes subcutaneous administration, intraperitoneal administration, oral administration, intradermal administration, intraventricular administration and the like.
  • “Increased in a development-dependent manner” means that the administration of the drug does not increase the expression level in the brain during infancy, but the expression level in the brain increases when the drug is administered during the maturation period. means.
  • the number and position of amino acids deleted, substituted, or added to the amino acid sequence of SEQ ID NO: 2 or 6 are particularly limited as long as the basic brain expression level decreases in a development-dependent manner.
  • the number is one or more, preferably one or several, and the specific range is usually 1 to 5, preferably 1 to 3, and more preferably 1 to 2. It is.
  • the amino acid sequence of the protein (b) has at least 99%, preferably at least 99.3%, more preferably at least 99.5%, homology with the amino acid sequence of the protein (a).
  • the protein (b) preferably has a PDZ domain and a PX domain in order from the N-terminal side. That is, the number and position of amino acids deleted, substituted, or added to the amino acid sequence of SEQ ID NO: 2 or 6 are within the range in which the PDZ domain and PX domain of the protein (a) are retained. Is preferred.
  • the number and position of amino acids deleted, substituted, or added to the amino acid sequence of SEQ ID NO: 4 or 8 are not particularly limited as long as the expression level in the brain decreases in a development-dependent manner.
  • the number is one or more, preferably one or several, and the specific range is usually 1 to 5, preferably 1 to 3, and more preferably 1 to 2.
  • the amino acid sequence of protein (d) is It has homology of usually at least 99%, preferably at least 99.3%, and more preferably at least 99.5% with the amino acid sequence of
  • the protein (d) preferably has a PDZ domain and a PX domain in order from the N-terminal side. That is, the number and position of amino acids deleted, substituted or added to the amino acid sequence of SEQ ID NO: 4 or 8 may be within the range in which the PDZ domain and PX domain of the protein (c) are retained. preferable.
  • the protein (d) preferably has the property that the expression level in the brain is increased in a brain-dependent manner by administration of a drug that causes a schizophrenia-like abnormality. That is, the number and position of amino acids deleted, substituted, or added to the amino acid sequence of SEQ ID NO: 4 or 8 are dependent on the expression level in the brain by administration of a drug that causes schizophrenia-like abnormalities. It is preferable that the temperature be within a range in which the property of increasing the temperature is maintained.
  • the protein (b) or (d) includes, in addition to a protein (a) or (c) into which a mutation such as deletion, substitution, or addition has been artificially introduced, as well as deletion, substitution, addition, etc.
  • proteins that naturally exist in a state in which a mutation has been introduced and proteins into which a mutation such as deletion, substitution, or addition has been artificially introduced.
  • Naturally occurring proteins in which mutations such as deletion, substitution, and addition have been introduced include, for example, mammals including humans (eg, humans, monkeys, horses, sheep, sheep, goats, horses, horses). And proteins (including proteins that can be caused by polymorphism in these mammals) from Escherichia coli, rabbits, dogs, cats, mice, rats and the like. .
  • the proteins (a), (b), (c) and (d) include both proteins with sugar chains and proteins without sugar chains.
  • the type, position, etc. of the sugar chains added to the protein vary depending on the type of host cell used in the production of the protein. Protein also includes proteins obtained using any host cells.
  • the protein (a), (b), (c) or (d) also includes a pharmaceutically acceptable salt thereof.
  • Genes encoding proteins (a), (b), (c) or (d) can be obtained, for example, from tissues of mammalian brain, heart, lung, spleen, liver, small intestine, testis, kidney, etc.
  • a cDNA library was prepared, and the desired DNA was obtained from the cDNA library using a probe synthesized based on the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 7. It is obtained by screening the containing clone.
  • each step of preparing a cDNA library and screening a clone containing the desired DNA will be described.
  • RNA is obtained by an affinity column method using poly U-Sepharose, a batch method, or the like.
  • poly (A +) RNA (mRNA) may be fractionated by sucrose gradient centrifugation or the like.
  • a single-stranded cDNA is synthesized using an oligo dT primer and a reverse transcriptase, and then a double-stranded cDNA is synthesized from the single-stranded cDNA. .
  • the thus obtained double-stranded cDNA is inserted into an appropriate cloning vector to prepare a recombinant vector, and a host cell such as Escherichia coli is transformed using the recombinant vector, and tetracycline resistance and ampicillin resistance are obtained.
  • a cDNA library can be obtained by selecting a transformant using as an index.
  • Cloning vectors for preparing a cDNA library may be those capable of autonomous replication in a host cell, such as a phage vector, A plasmid vector or the like can be used.
  • a host cell such as a phage vector, A plasmid vector or the like can be used.
  • the host cell for example, Escherichia coli and the like can be used.
  • Transformation of a host cell such as Escherichia coli can be performed by a method of adding a recombinant vector to a competent cell prepared in the presence of calcium chloride, magnesium chloride, or rubidium chloride.
  • plasmid When plasmid is used as a vector, it is preferable to contain a drug resistance gene such as tetracycline or ampicillin.
  • kits such as SuperScript Plasiid System for cDNA Synthesis and Plasmid Cloning (manufactured by Gibco BRL) and ZAP-cDNA Synthesis Kit (manufactured by Stratagene) can be used.
  • PCR PCR amplified fragment
  • the PCR amplified fragment may be subcloned using an appropriate plasmid vector.
  • the set of primers used for PCR is not particularly limited, and can be designed based on the nucleotide sequence described in SEQ ID NO: 1, 3, 5, or 7.
  • the target DNA is obtained by performing colony hybridization or plaque hybridization on the cDNA library using the PCR amplified fragment as a probe.
  • a PCR amplified fragment labeled with an isotope for example, 32 P, 35 S
  • biotin for example, 32 P, 35 S
  • digoxigenin for example, digoxigenin
  • alkaline phosphatase or the like
  • a clone containing the desired DNA can also be obtained by expression screening such as immunoscreening using an antibody.
  • the nucleotide sequence of the obtained DNA is obtained by digesting the DNA fragment as it is or after digestion with an appropriate restriction enzyme or the like, incorporating the DNA fragment into a vector by a conventional method, and using a commonly used nucleotide sequence analysis method, for example, chemical modification of Maxam-Gilbert. Method, dideoxynucleotide chain termination.
  • a nucleotide sequence analyzer such as 3773 DNA Sequencer (Perkin Elmer) is usually used.
  • the gene encoding protein (a), (b), (c) or (d) contains an open reading frame and a stop codon located at its 3 'end.
  • the gene encoding the protein (a), (b), (c) or (d) contains an untranslated region (UTR) at the 5 ′ end and at the 3 ′ end of the open reading frame. be able to.
  • Examples of the gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 include a gene containing DNA consisting of the nucleotide sequence of the 25th to 1847th nucleotides in the nucleotide sequence of SEQ ID NO: 1.
  • the open reading frame is located at the nucleotide sequence at positions 225-1847
  • the translation initiation codon is located at the nucleotide sequence at positions 225-227
  • the termination is indicated.
  • the codon is located at the nucleotide sequence at positions 1848-185.
  • the nucleotide sequence of the gene encoding the protein consisting of the amino acid sequence of SEQ ID NO: 2 is not particularly limited as long as it encodes the protein, and the nucleotide sequence of the open reading frame is as described in SEQ ID NO: 1.
  • the nucleotide sequence is not limited to the nucleotide sequence at positions 225-1847.
  • Examples of a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4 include a gene containing DNA consisting of the nucleotide sequence of the 25th to 180th nucleotides in the nucleotide sequence of SEQ ID NO: 3. No. Here, of the nucleotide sequence described in SEQ ID NO: 3, the open reading frame is 2 The translation start codon is located at the 25th to 227th nucleotide sequence, and the stop codon is located at the 180th to 181st nucleotide sequence. I do.
  • nucleotide sequence of the gene encoding the protein consisting of the amino acid sequence of SEQ ID NO: 4 is not particularly limited as long as it encodes the protein, and the nucleotide sequence of the open reading frame is the nucleotide sequence of SEQ ID NO: 3.
  • the present invention is not limited to the base sequence of the 25th to 180th bases.
  • Examples of the gene encoding the protein consisting of the amino acid sequence of SEQ ID NO: 6 include a gene containing a DNA consisting of the nucleotide sequence of the 121st to 137th nucleotides in the nucleotide sequence of SEQ ID NO: 5. .
  • the open reading frame is located at the nucleotide sequence at positions 121 to 173
  • the translation initiation codon is located at the nucleotide sequence at positions 121 to 123.
  • the stop codon is located at the nucleotide sequence at positions 1738 to 1740.
  • the nucleotide sequence of the gene encoding the protein consisting of the amino acid sequence of SEQ ID NO: 6 is not particularly limited as long as it encodes the protein, and the nucleotide sequence of the open reading frame is the nucleotide sequence of SEQ ID NO: 5. Of these, it is not limited to the 121st to 1737th base sequence.
  • Examples of the gene encoding the protein consisting of the amino acid sequence of SEQ ID NO: 8 include a gene containing a DNA consisting of the nucleotide sequence at positions 121 to 169 of the nucleotide sequence of SEQ ID NO: 7. .
  • the open reading frame is located at the nucleotide sequence of 121 to 169
  • the translation initiation codon is located at the nucleotide sequence of 121 to 123.
  • the stop codon is located in the base sequence at position 169 to 1701.
  • the nucleotide sequence of the gene coding for the protein consisting of the amino acid sequence of SEQ ID NO: 8 may be any There is no particular limitation, and the nucleotide sequence of the open reading frame is not limited to the nucleotide sequence at positions 121 to 168 of the nucleotide sequence shown in SEQ ID NO: 7.
  • the gene encoding the protein (a), (b), (c) or (d) can also be obtained by chemical synthesis according to its nucleotide sequence.
  • Chemical synthesis of DNA is carried out using a commercially available DNA synthesizer, such as a DNA synthesizer using the thiophosphite method (manufactured by Shimadzu Corporation) and a DNA synthesizer using the phosphoramidite method (manufactured by Perkin Elmer Inc.) ).
  • Examples of the gene encoding the protein (b) or (d) include, for example, the nucleotide sequences of SEQ ID NOS: 1, 3, 5, and 7 at positions 225-1847, and 225-18008, respectively.
  • the “stringent conditions” include, for example, the conditions of 42 ° C, 2 XSSC and 0.1% SDS, preferably 65 ° (: 0.1, 33 ° and 0.1% SDS. No.
  • the DNA that hybridizes to the DNA complementary to the DNA consisting of the base sequence at position 8 under stringent conditions includes, among the base sequences of SEQ ID NOs: 1, 3, 5, and 7, 1847th, 225th to 188th, 1211 to 1737th or 1211 to 169th DNA consisting of the base sequence and at least 95% or more, preferably 9 or more
  • a DNA having a homology of 7% or more, more preferably 98% or more, may be mentioned.
  • the gene encoding the protein (b) or (d) can be obtained by artificially mutating the gene encoding the protein (a) or (c) using a known method such as site-directed mutagenesis. It can also be obtained by introducing. Mutagenesis can be introduced using, for example, a mutagenesis kit, for example, Mutant-K (manufactured by TAKARA), Mutant-G (manufactured by TAKARA), or TAKARA's LA PCR in vitro Mutagenesis series kit. it can. In addition, a gene whose base sequence has already been determined can be obtained by chemical synthesis according to the base sequence.
  • the protein (a), (b), (c) or (d) can be produced, for example, by expressing a gene encoding each protein in a host cell according to the following steps.
  • DNA fragment of an appropriate length containing the coding region of the target protein is prepared.
  • DNA is prepared by substituting the base so that the base sequence of the coding region of the target protein is an optimal codon for expression in a host cell.
  • a recombinant vector is prepared by inserting this DNA fragment downstream of the promoter of an appropriate expression vector, and the recombinant protein is introduced into an appropriate host cell to produce a desired protein. The resulting transformant is obtained.
  • the above-mentioned DNA fragment must be integrated into the vector so that its function can be exhibited.
  • the vector may be a cis element such as an enhancer, a splicing signal, a poly-A addition signal, or the like. It can contain a selectable marker (eg, a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene), a ribosome binding sequence (SD sequence), and the like.
  • Expression vectors are capable of autonomous replication in a host cell.
  • a plasmid vector a phage vector, a virus vector, or the like.
  • the plasmid vector include plasmids derived from Escherichia coli (eg, pRSET, pBR322, pBR325, pUCll8, pUC119, pUC18, pUC19), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5), and plasmids derived from yeast ( Examples of phage vectors include ⁇ phage (eg, Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gtlO AgtlK ⁇ ZAP), and examples of virus vectors include: Examples include animal viruses such as retroviruses and vaccinia viruses, and insect viruses such as baculoviruses.
  • any of prokaryotic cells, yeast, animal cells, insect cells, plant cells, etc. may be used as long as the target gene can be expressed.
  • animal individuals, plant individuals, silkworms, etc. may be used.
  • a bacterium When a bacterium is used as a host cell, for example, genus Escherichia such as Escherichia coli, genus Bacillus such as Bacillus s subtilis, genus Pseudomonas putida such as Pseudomonas put ida, etc. ⁇ Bacteria belonging to the genus Rhizobium, such as meliloti (Rhizobium mel i lot i), can be used as host cells.
  • Rhizobium such as meliloti (Rhizobium mel i lot i)
  • Escherichia coli BL2K Escherichia coli i XLl-Blue, Escherichia coli XL2-Blue> Escherichia coli DH1, Escherichia coli Kl2, Escherichia coli JM109 Escherichia coli HB101, etc., and Bacillus subtilis MI114 Bacillus subtilis 207-21 and the like can be used as host cells.
  • Promoter one in this case is not particularly limited as long as it can be expressed in bacteria such as Escherichia coli, for example, trp promoter one, c promoter, P L promoter, the P R promoter Isseki one like Escherichia coli or phage, etc.
  • Derived promoters can be used.
  • tac promo -Artificially designed and modified promoters, such as lacT7 promoter, let I promoter, etc. can also be used.
  • the method for introducing the recombinant vector into the bacterium is not particularly limited as long as it is a method capable of introducing the DNA into the bacterium.
  • a method using a calcium ion, an electrolysis method and the like can be used.
  • yeast When yeast is used as a host cell, Saccharomyces cerevisiae. Schizosaccharomyce s pombe, Pichia pastoris, and the like can be used as host cells.
  • the promoter in this case is not particularly limited as long as it can be expressed in yeast.
  • gall promoter, gal 10 promoter, heat shock protein promoter, MFal promoter, PH05 promoter, PGK promoter , GAP promoter, ADH promoter, A0X1 promoter, etc. can be used.
  • the method for introducing the recombinant vector into yeast is not particularly limited, as long as it is a method capable of introducing DNA into yeast.
  • a method capable of introducing DNA into yeast For example, an elect-portation method, a spheroplast method, a lithium acetate method, or the like can be used. .
  • monkey cells such as COS-7, Vero, Chinese hamster ovary cells (CH0 cells), mouse L cells, rat GH3, and human FL cells can be used as host cells.
  • the promoter in this case is not particularly limited as long as it can be expressed in animal cells.
  • SRa promoter SV40 promoter, LTR (Long Terminal Repeat) promoter, CMV promoter, early gene promoter of human cytomegalovirus First class can be used.
  • the method for introducing the recombinant vector into animal cells is not particularly limited, as long as it is a method capable of introducing DNA into animal cells.
  • an electoporation method, a calcium phosphate method, a Lipofection method and the like can be used.
  • an insect cell is used as a host, an ovary cell of Spodoptera frugiperda, an ovary cell of Trichoplusia ni, a cultured cell derived from a silkworm ovary, or the like can be used as a host cell. Spod 9, S111, etc.
  • the method for introducing the recombinant vector into the insect cells is not particularly limited as long as the DNA can be introduced into the insect cells.
  • a calcium phosphate method, a ribofection method, an electrolysis method and the like can be used.
  • the transformant into which the recombinant vector incorporating DNA encoding the protein of interest has been introduced is cultured according to a conventional culture method.
  • the transformant can be cultured according to a conventional method used for culturing host cells.
  • a medium for culturing a transformant obtained by using a microorganism such as Escherichia coli or yeast as a host cell contains a carbon source, a nitrogen source, inorganic salts, and the like, which can be used by the microorganism, to efficiently culture the transformant.
  • a natural medium or a synthetic medium may be used as long as the medium can be used for the above.
  • the carbon source carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used.
  • the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other inorganic or organic acid ammonium salts, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, etc.
  • Inorganic salts include potassium (II) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and sulfuric acid.
  • Manganese oxide, copper sulfate, calcium carbonate, etc. can be used.
  • Culture of a transformant obtained by using a microorganism such as Escherichia coli or yeast as a host cell is performed under aerobic conditions such as shaking culture or aeration and stirring culture.
  • the culture temperature is usually 25 to 37 ° C
  • the culture time is usually 12 to 48 hours
  • the pH is maintained at 6 to 8 during the culture period.
  • the pH can be adjusted using an inorganic acid, an organic acid, an alkali solution, urea, calcium carbonate, ammonia, or the like.
  • an antibacterial substance such as ampicillin or tetracycline may be added to the medium as needed. .
  • an inducer may be added to the medium, if necessary.
  • an inducer may be added to the medium, if necessary.
  • isobutyl-i3-D-thiogalactopyranoside and the like can be used to culture a microorganism transformed with an expression vector using the trp promoter.
  • indole acrylic acid or the like may be added to the medium.
  • a medium for culturing the transformant obtained using animal cells as host cells commonly used RPMI 1640 medium, Eagle's MEM medium, DMEM medium, Ham F12 medium, Ham F12K medium or a bovine fetal medium A medium containing serum or the like can be used. Culture of the transformant is carried out normally in 5% C_ ⁇ 2 presence, 3 7 ° C in 3-1 0 days. At the time of culturing, if necessary, antibiotics such as kanamycin, penicillin, streptomycin and the like may be added to the medium.
  • a medium for culturing a transformant obtained by using an insect cell as a host cell commonly used T-band FH medium (Pharmingen), Sf-900 II SFM medium (Gibco BRL) , ExCel OO, ExCell405 URH Biosciences) can be used. Culture of the transformant is usually performed at 27 ° C. for 3 to 10 days. When culturing, if necessary, use gentamicin, etc. May be added to the medium.
  • the target protein can also be expressed as a secreted protein or a fusion protein.
  • proteins to be fused are: / 3 monogalactosidase, protein A, IgG binding region of protein A, chloramphenicol acetyltransferase, poly (Arg), poly (G1u), protein G, maltose Binding protein, dalzygosine S-transferase, polyhistidine chain (His-tag), S peptide, DNA binding protein domain, Tac antigen, thioredoxin, Darine fluorescens ⁇ protein and the like.
  • the target protein By collecting the target protein from the culture of the transformant, the target protein can be obtained.
  • the “culture” includes any of culture supernatant, cultured cells, cultured cells, and crushed cells or cells.
  • the target protein When the target protein is accumulated in the cells of the transformant, the cells in the culture are collected by centrifuging the culture, and the cells are washed, and then the cells are disrupted. The protein to be extracted is extracted.
  • the target protein When the target protein is secreted out of the cells of the transformant, use the culture supernatant as it is, or remove cells or cells from the culture supernatant by centrifugation or the like.
  • the protein (a), (b), (c) or (d) thus obtained is obtained by solvent extraction, salting out using ammonium sulfate, desalting, precipitation with an organic solvent, getyl aminoethyl (DEAE) -cephalo It can be purified by ion-exchange chromatography, hydrophobic chromatography, gel filtration, affinity chromatography.
  • the protein (a;), (b), (c) or (d) is based on the Fmoc method (fluorenylmethyloxycarbonyl method), the tBoc method (t Monobutyloxycarbonyl method) and the like. In this case, a commercially available peptide synthesizer can be used.
  • the antibody or fragment thereof of the present invention is an antibody or a fragment thereof capable of reacting with the protein (a), (b), (c) or (d).
  • antibody includes both monoclonal antibodies and polyclonal antibodies, and “monoclonal antibodies and polyclonal antibodies” include all classes of monoclonal antibodies and polyclonal antibodies.
  • the term “antibody” includes antiserum obtained by immunizing immunized animals such as rabbits and mice with the protein (a), (b), (c) or (d), human antibodies, and those obtained by genetic recombination. Also includes humanized antibodies.
  • the “antibody fragment” includes Fab fragments, F (ab) ′ 2 fragments, single-chain antibodies (scFv) and the like.
  • the antibody or fragment thereof of the present invention can be prepared by using the protein (a), (b), (c) or (d) as an antigen for immunization.
  • the antigen for immunization include (i) a fragment of a cell or tissue expressing the protein (a), (b), (c) or (d) or a purified product thereof, and (ii) a genetic recombination technique.
  • a recombinant protein obtained by introducing a gene encoding the protein (a), (b), (c) or (d) into a host such as Escherichia coli, an insect cell or an animal cell and expressing the gene; Synthetic peptides and the like can be used.
  • a mammal such as a rat, a mouse, a guinea pig, a heron, a sheep, a poma, or a mosquito is immunized with an antigen for immunization.
  • an antigen for immunization it is preferable to use egrets because antibodies can be easily produced.
  • immunization in order to induce antibody production, it is preferable to carry out immunization a plurality of times after emulsification using an immune auxiliary such as Freund's complete adjuvant.
  • an immune auxiliary such as Freund's complete adjuvant.
  • Freund's complete adjuvant FCA
  • FIA incomplete Freund's adjuvant
  • aluminum hydroxide gel and the like can be used.
  • the dose of antigen per mammal is although it can be set appropriately according to the type of mammal, it is usually 200 to 200 ⁇ g in the case of a perch.
  • the administration site is, for example, intravenous, intradermal, subcutaneous, intraperitoneal, or the like.
  • the immunization is carried out usually at intervals of several days to several weeks, preferably at intervals of 5 days to 3 weeks, for a total of 3 to 10, preferably 5 to 7 immunizations. Then, 5 to 20 days after the last immunization, the antibody titer against the protein (a), (b), (c) or (d) is measured, and blood is collected after the antibody titer has risen. Get.
  • the antibody titer can be measured by enzyme immunoassay (ELISA), radioimmunoassay (RIA), or the like.
  • purification of the antibody from the antiserum is required, known methods such as salting out with ammonium sulfate, gel-mouth chromatography, ion-exchange chromatography, and affinity chromatography are appropriately selected or used. They can be used in combination.
  • a mammal is immunized with an immunizing antigen in the same manner as in the case of a polyclonal antibody, and antibody-producing cells are collected 5 to 20 days after the last immunization.
  • the antibody-producing cells include spleen cells, lymph node cells, thymocytes, peripheral blood cells, and the like, and spleen cells are generally used.
  • cell fusion between antibody-producing cells and myeloma cells is performed.
  • a cell line derived from a mammal such as human or mouse and generally available can be used as the myeoma cell to be fused with the antibody-producing cell.
  • the cell line to be used is preferably a cell line which has drug selectivity, cannot survive in a selection medium (for example, HAT medium) in an unfused state, and can survive only in a state fused to an antibody-producing cell.
  • myeloma cells include mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1) P3 / NSI / 1-Ag4-1, Sp2 / 0-Agl4.
  • a cell fusion reaction animal cells cultured in serum-free DMEM, RPMI-1640 medium, etc.
  • Antibody-producing cells and myeloma cells are mixed at a predetermined ratio (for example, 5: 1 to 20: 1) in a culture medium for use in the presence of a cell fusion promoter such as polyethylene glycol, or by electric pulse treatment (for example, The fusion reaction is performed by electroporation.
  • a cell fusion promoter such as polyethylene glycol
  • the cells are cultured using a selection medium to select the desired hybridoma.
  • screening is performed to determine whether or not the target antibody is present in the culture supernatant of the grown hybridoma. Screening for hybridomas may be performed according to a conventional method, and is not particularly limited. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by an enzyme immunoassay (ELISA), a radioimmunoassay (RIA), or the like.
  • ELISA enzyme immunoassay
  • RIA radioimmunoassay
  • the cloning of the hybridoma can be performed by, for example, the limiting dilution method, the soft agar method, the Fibrin gel method, the fluorescence-excited Celso overnight method, etc., and finally obtain the hybridoma that produces the monoclonal antibody.
  • an ordinary cell culture method or the like can be used as a method for collecting a monoclonal antibody from the obtained hybridoma.
  • hybridomas are cultured in animal cell culture medium such as RPMI-1640 medium containing 10 to 20% fetal bovine serum, MEM medium, etc. under normal culture conditions (for example, 37 ° C, 5% CO2).
  • a monoclonal antibody By culturing at 2 concentrations) for 3 to 10 days, a monoclonal antibody can be obtained from the culture supernatant.
  • the hybridoma can be transplanted into the abdominal cavity of a mouse or the like, and ascites can be collected 5 to 20 days later, and a monoclonal antibody can be obtained from the ascites.
  • the monoclonal antibody When purification of the monoclonal antibody is required, known methods such as salting out with ammonium sulfate, gel-closing chromatography, ion-exchange chromatography, affinity chromatography, etc. are appropriately selected or used. Available in combination.
  • a human antibody or a humanized antibody When the monoclonal antibody is used for the purpose of administration to humans (antibody therapy), it is preferable to use a human antibody or a humanized antibody in order to reduce immunogenicity.
  • Human antibody or humanized antibody can be obtained, for example, by producing a hybridoma using a mouse or the like into which a human antibody gene has been introduced as an immunized animal, or by using a library displaying the antibody on phage. it can.
  • a transgenic animal having a repertoire of human antibody genes is immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain antibody-producing cells, which are then fused with myeloma cells.
  • a human antibody against the target protein can be obtained using the hybridomas (see International Publication Nos. W092-03918, W093-227, W094-02602, W096-33735 and W096-34096).
  • a phage presenting an antibody that binds to a protein serving as an antigen a protein-expressing cell or a lysate thereof from an antibody library in which a plurality of different human scFvs are displayed on the phage
  • ScFvs that bind to the protein of interest can be selected (Griffit hs. Et al., EMBO J. 12, 725-734, 1993).
  • the diagnostic method of the present invention provides a method for determining whether a test animal is suffering from schizophrenia or a schizophrenia by indexing the expression level of the gene encoding protein (c) or (d) in a brain sample collected from the test animal. It includes the step of diagnosing whether or not an abnormality is exhibited as a disease model.
  • the gene encoding the protein (c) or (d) is not upregulated in the brain of a healthy animal that has transitioned from immature to maturity or has reached maturity, but is suffering from schizophrenia
  • the expression level of the gene is up-regulated in the schizophrenia or schizophrenia-like abnormalities because the expression level of the gene is upregulated in the brain of humans or in the brains of humans and non-human animals exhibiting abnormalities as a schizophrenia model. It can be used as an index for diagnosis.
  • test animals are not particularly limited, and include, for example, humans, monkeys, sea lions, sheep, goats, horses, horses, cats, dogs, cats, rats, mice, etc. Mammals. Since schizophrenia or an abnormality as a schizophrenia model is manifested after the transition from juvenile to maturity or after reaching maturity, the test animals should be in the transition from juvenile to maturity Alternatively, animals that have reached maturity are used. Brain specimens collected from test animals include various parts of the brain such as cerebral neocortex, cerebral neocortex, striatum, hippocampus, cerebellum, limbic forebrain, thalamus, hypothalamus, midbrain, brainstem, etc. Parts or mixtures of these can be used.
  • the “expression level of the gene encoding the protein (c) or (d)” includes the level of transcription of the gene encoding the protein (c) or (d) into mRNA and the level of transcription into the protein (c) or (d). Translation level is included. Therefore, the expression level of the gene encoding the protein (c) or (d) in the brain sample is determined by the abundance of the mRNA encoding the protein (c) or (d) in the brain sample or the protein (b) in the brain sample. It can be measured based on the abundance of c) or (d).
  • a known gene analysis technique for example, an eighty hybridization technique (for example, the Northern hybridization method, Blotting, DNA microarray, etc.), gene amplification technology (eg, RT-PCR, etc.) and the like.
  • an oligonucleotide or polynucleotide capable of hybridizing to the nucleic acid encoding the protein (c) or (d) can be used as a probe.
  • Nucleic acid encoding protein (c) or (d) includes both DNA and RNA, and includes, for example, mRNA, cDNA, cRNA, etc. It is.
  • the nucleotide constituting the oligonucleotide or the polynucleotide may be any of deoxyliponucleotide and liponucleotide.
  • the base length of the oligonucleotide is not particularly limited, but is usually 20 to 100 bases, and preferably 20 to 40 bases. Further, the base length of the polynucleotide is not particularly limited, but is usually 300 to 200 bases, preferably 700 to 100 bases.
  • Oligonucleotides or polynucleotides capable of hybridizing to the nucleic acid encoding the protein (c) or (d) are preferably capable of specifically hybridizing to the nucleic acid encoding the protein (c) or (d).
  • “Specifically hybridizable” means that it can hybridize under stringent conditions.
  • Stringent conditions include, for example, 42 ° C, 2 XSSC and 0.1% SDS. , Preferably at 65 ° C., 0.1% 33 ° and 0.1% SDS.
  • the nucleotide sequence of an oligonucleotide or a polynucleotide capable of hybridizing to the nucleic acid encoding the protein (c) or (d) can be designed based on the nucleotide sequence of the nucleic acid encoding the protein (c) or (d).
  • the oligonucleotide or polynucleotide is hybridized to the 5'-terminal or 3'-terminal region of the CDS region, for example, so that it can hybridize to the CDS region of the nucleic acid encoding the protein (c) or (d).
  • a restriction enzyme recognition sequence, a tag, and the like can be added to the 5 'end of the primer, and a label such as a fluorescent dye or a radioisotope can be added to the primer and the probe.
  • a specific method for measuring the abundance of the mRNA encoding the protein (c) or (d) in the brain sample will be described using an example in which RT-PCR is used.
  • RNA extract and extract total RNA from specimens collected from test animals After synthesizing cDNA from all the RNAs obtained, the synthesized cDNA was converted into type III, and PCR was performed using a primer capable of hybridizing to cDNA encoding protein (c) or (d), and By quantifying the PCR amplified fragment, the abundance of mRNA encoding the protein (c) or (d) can be measured. At this time, PCR is performed under conditions such that the amount of PCR-amplified fragment generation reflects the amount of the initial type III cDNA (for example, the number of PCR cycles at which the PCR-amplified fragment increases exponentially).
  • the method for quantifying the PCR amplified fragment is not particularly limited, and the PCR amplified fragment can be quantified by, for example, a quantification method using a radioisotope (RI), a quantification method using a fluorescent dye, or the like.
  • RI radioisotope
  • Examples of the quantification method using RI include: (i) adding a RI-labeled nucleotide (for example, 32 P-labeled dCTP, etc.) as a substrate to a reaction solution, incorporating the nucleotide into an amplified PCR fragment, and PCR amplification.
  • RI-labeled primers A method in which the amplified PCR fragment is labeled with RI, the amplified PCR fragment is separated by electrophoresis and the like, and the radioactivity is measured to quantify the amplified PCR fragment.
  • electrophoresis of the amplified PCR fragment A method of quantifying the PCR-amplified fragment by measuring the radioactivity by hybridizing the probe labeled with RI, and measuring the radioactivity. The radioactivity can be measured using, for example, a liquid scintillation counter, an X-ray film, an imaging plate, or the like.
  • Quantitative methods using fluorescent dyes include (i) PCR amplification fragments using a fluorescent dye that interacts with double-stranded DNA (eg, ethidium bromide (EtBr), SYBR GreenI, coGreen, etc.).
  • a method for quantifying PCR amplified fragments by staining and measuring the fluorescence intensity emitted by excitation light irradiation (ii) The PCR-amplified fragment is labeled with a fluorescent dye by using a primer labeled with a fluorescent dye, the PCR-amplified fragment is separated by electrophoresis, etc., and the fluorescence intensity is measured to determine the PCR-amplified fragment.
  • the fluorescence intensity can be measured using, for example, a CCD camera, a fluorescence scanner, a spectrofluorometer, or the like.
  • known protein analysis techniques for example, Western blotting using an antibody or a fragment thereof capable of reacting with the protein (c) or (d) are used. Plotting, immunoprecipitation, ELISA, tissue immunostaining, etc. can be used.
  • the protein (c) or (d) in a sample using an antibody or a fragment thereof that can react with the protein (c) or (d) for example, radioimmunoassay (RIA) , Enzyme immunoassay (EIA), chemiluminescence assay (CLIA), fluorescence immunoassay (FIA), tissue immunostaining, etc. can be used.
  • RIA radioimmunoassay
  • EIA Enzyme immunoassay
  • CLIA chemiluminescence assay
  • FIA fluorescence immunoassay
  • tissue immunostaining etc.
  • the protein (c) or (d) in the sample is captured using a solid-phase carrier (eg, immunoplate, latex particles, etc.) to which the antibody is bound by physical adsorption or chemical bonding.
  • a solid-phase carrier eg, immunoplate, latex particles, etc.
  • the captured protein (c) or (d) is labeled with a labeled antibody (for example, peroxidase, aloxidase) having a different antigen-recognition site for the protein (c) or (d) from the antibody immobilized on the solid support.
  • a labeled antibody for example, peroxidase, aloxidase
  • an antibody labeled with a fluorescent substance such as fluorescens and umberiferon).
  • the measurement of the amount of the protein (c) or (d) in the brain sample can be performed by measuring the activity of the protein (c) or (d) in the brain sample.
  • the activity of the protein (c) or (d) can be measured, for example, by a known method such as a Western plotting method or an ELISA method using an antibody or a fragment thereof capable of reacting with the protein (c) or (d).
  • the measured value of the expression level of the protein (c) or (d) is preferably corrected based on the measured value of the expression level of a protein (eg,
  • the expression level of the gene encoding the protein (c) or (d) in the brain sample collected from the test animal is determined by the expression level of the protein (c) or (d) in the brain sample collected from a healthy animal.
  • the test animal can be diagnosed as having schizophrenia or exhibiting an abnormality as a schizophrenia model.
  • the expression level of the gene encoding protein (c) or (d) between a test animal and a healthy animal it is preferable to use the same site or part of the brain as the specimen.
  • the expression levels in a plurality of healthy animals (healthy animal groups) were quantified. It is preferable to set a normal range from the distribution of the values and determine whether the expression level in the test animal is higher than or lower than the normal range. At this time, when the expression level of the gene in the brain specimen of the test animal is above the normal range, it can be diagnosed that the test animal is suffering from schizophrenia or exhibiting an abnormality as a schizophrenia model.
  • the diagnostic kit of the present invention comprises an oligonucleotide or polynucleotide capable of hybridizing to a nucleic acid encoding the protein (c) or (d), an antibody capable of reacting with the protein (c) or (d), or a fragment thereof. Including. These oligonucleotides, polynucleotides, antibodies or fragments thereof are used as reagents for measuring the expression level of the gene encoding protein (c) or (d) in a brain sample collected from a test animal, and are used as diagnostic reagents of the present invention. Kit, and uses the diagnostic kit of the present invention. This makes it possible to diagnose whether the test animal is suffering from schizophrenia or exhibiting abnormalities as a schizophrenia model.
  • the diagnostic kit of the present invention may be in any form as long as it contains the above-mentioned oligonucleotide or polynucleotide, or the above-mentioned antibody or fragment thereof, and may contain any reagents, instruments and the like.
  • the diagnostic kit of the present invention contains the above-mentioned oligonucleotide or polynucleotide, reagents necessary for PCR (for example, H 2 ⁇ , buffer, MgCl 2 , dNTP mix, TaQ polymerase, etc.) It can contain one or more of the reagents required for quantification of PCR amplified fragments (eg, RI, fluorescent dye, etc.), DNA microarray, DNA chip, etc.
  • reagents necessary for PCR for example, H 2 ⁇ , buffer, MgCl 2 , dNTP mix, TaQ polymerase, etc.
  • the diagnostic kit of the present invention contains the above antibody or a fragment thereof, a solid phase carrier for immobilizing the antibody or the fragment thereof (for example, immunoplate, latex particles, etc.), anti-agrodarin One type of antibody (secondary antibody), labeling of antibody (including secondary antibody) or a fragment thereof (eg, enzyme, fluorescent substance, etc.), various reagents (eg, enzyme substrate, buffer, diluent, etc.) or Two or more types can be included.
  • a solid phase carrier for immobilizing the antibody or the fragment thereof for example, immunoplate, latex particles, etc.
  • anti-agrodarin One type of antibody secondary antibody
  • labeling of antibody including secondary antibody
  • a fragment thereof eg, enzyme, fluorescent substance, etc.
  • various reagents eg, enzyme substrate, buffer, diluent, etc.
  • Two or more types can be included.
  • the screening method of the present invention comprises the steps of integrating the expression of a gene encoding a protein (c) or (d) into cells or tissues (including organs) in which the expression level of the gene is increased, using the effect of reducing the expression level of the gene as an index. It includes the step of determining the preventive and therapeutic effects of the candidate substance on schizophrenia or schizophrenia-like abnormalities. Since schizophrenia or schizophrenia-like abnormalities is associated with enhanced expression of the gene encoding protein (c) or (d), expression of the gene encoding protein (c) or (d) By selecting a substance having a level reducing effect, it is possible to screen a substance for preventing and treating schizophrenia or schizophrenia-like abnormalities.
  • the “progress” refers to a state in which the gene is introduced as a foreign gene and forced expression, a state in which the expression level of the gene inherent to the host is increased, and the degradation of the protein (c) or (d) is suppressed. All of the states that have been performed are included.
  • the “effect of reducing the expression level of the gene encoding the protein (c) or (d)” includes the transcription and translation of the gene encoding the protein (c) or (d), and the protein (c) or (d). The effect on any step, such as the expression of the activity of the enzyme, is also included.
  • the screening method of the present invention can be performed both in vivo and invitro.
  • a specimen before administration of the candidate substance, Cells or tissues with elevated expression levels of the gene (e.g., brain) are collected, and schizophrenia or integration is determined based on whether or not the expression level of the gene has been reduced in the specimen.
  • schizophrenia or integration is determined based on whether or not the expression level of the gene has been reduced in the specimen.
  • the model animals used in the screening method of the present invention include, for example, mammals such as porcupine, sheep, goat, porcupine, pig, porcupine, dog, cat, rat, and mouse. .
  • mammals such as porcupine, sheep, goat, porcupine, pig, porcupine, dog, cat, rat, and mouse.
  • a model animal in which the expression level of the gene encoding the protein (c) or (d) is elevated a model animal exhibiting schizophrenia-like abnormalities can be used as a model animal in which the expression level of the gene encoding the protein (c) or (d) is elevated.
  • Model animals showing schizophrenia-like abnormalities include drugs that cause schizophrenia-like abnormalities, for example, stimulants such as amphetamine and methamphetamine or derivatives thereof; central stimuli such as cocaine, methylphenidite or derivatives thereof.
  • Dopamine agonists such as drugs; D0PA, promocributin, apomorphine, etc. Can be prepared.
  • transgenic animals in which the expression level of the gene encoding the protein (C) or (d) is artificially enhanced can be used.
  • Such transgenic animals can be obtained, for example, by (i) a method of mixing a gene encoding the protein (c) or (d) with an egg and treating it with calcium phosphate, (ii) a pronuclear phase under a phase-contrast microscope. It is obtained by a known method such as a method of directly introducing a gene encoding the protein (c) or (d) into the nucleus of the egg (microinjection method), and (iii) a method using embryonic stem cells (ES cells). be able to.
  • Cells used in the in vitro screening method include, for example, cell lines derived from humans, monkeys, mice, rats, etc. (for example, PC12, NB9, NB69, C-1300, COS-7, CH0, GH3, FL, L) are available.
  • cells in which the expression level of the gene encoding the protein (c) or (d) is artificially enhanced can also be used.
  • Such cells can be obtained by inserting a gene encoding the protein (c) or (d) into an appropriate expression vector, and introducing the vector into an appropriate host cell.
  • the screening kit of the present invention comprises an oligonucleotide or polynucleotide capable of hybridizing to a nucleic acid encoding the protein (c) or (d). Nucleotide, or an antibody or a fragment thereof capable of reacting with the protein (c) or (d). These oligonucleotides, polynucleotides, antibodies or fragments thereof are used as a reagent of the present invention as a reagent for measuring the expression level of the gene encoding protein (c) or (d) in a sample collected from a test animal.
  • the prophylactic / therapeutic substance for schizophrenia or schizophrenia-like abnormalities can be screened by using the screening kit of the present invention, which is included in a kit for learning.
  • the screening kit of the present invention may be in any form as long as it contains the above-mentioned oligonucleotide or polynucleotide, or the above-mentioned antibody or fragment thereof. It can include a substance, a candidate substance synthesis kit, a model animal breeding kit, and the like.
  • the present invention will be described in detail based on examples.
  • mrtl represents a gene
  • Mrtl represents a protein
  • MAP hydrochloride, co-in hydrochloride and SCH23390 hydrochloride were dissolved in physiological saline and used for subcutaneous (s.c.) or intraperitoneal (i.p.) administration.
  • the ampoule solution of pentobarbital was diluted with saline and used for i.p. administration. Control animals received vehicle only. Dosage always refers to the free principal component.
  • RNA fingerprinting (RNA arbitrarily primed PGR (RAP-PCR)) using any 12-mer primer to isolate developmentally dependent neocortical MAP responsive transcripts (Welsh J Et al., Nucleic Acids Res, 1992, 20, 4965-4970). 8 or 50 day old rats were given 0.8 kg / k of MAP or saline s., And killed 1 hour later by cervical dislocation. (1) The purpose of this study was to gain insight into the early or early stages of the putative molecular cascade of behavioral sensitization (2) 4.8 mg / kg MAP. After single or repeated treatment with, a sensitization phenomenon was observed in early adult rats, but not in young rats. Therefore, the dose and the timing of MAP administration were selected.
  • RNA Preamplification Kit RNA Preamplification Kit
  • PCR was performed using an arbitrary 12-mer primer (5'-caggtgggacca-3 '). PCR products were separated on a 4% denaturing polyacrylamide gel containing 50% urea. Gels are treated with SYBR Green I (Molec cDNA bands were visualized and analyzed with a Fluorlmager SI Fluo resceni image analyzer (Molecular Dynamics).
  • RNA levels in each sample was performed by RT-PCR, which co-amplifies the specific target sequence with other endogenous or competitive sequences that are internal standards (Foley KP et al., Trends Genet, 1993). , 9, 380-385).
  • Reverse transcription was performed using 0.2 g of total RNA obtained from the neocortex of each rat.
  • the obtained cDNA was suspended in 10 volumes of TE buffer (PH8.0), and the IL aliquot of the cDNA suspension was used as a type II PCR.
  • the 3 'end of 73% of the 18S rRNA primer was phosphorylated and 27 cycles of PCR amplification (45 seconds at 94 ° C, 45 seconds at 60 ° C, 45 seconds at 72 ° C) was defined as one cycle) (see Fig. 3C).
  • the same aliquot of the cDNA is used to dilute the competitive sequence that is identical to the target sequence except for the point mutation that creates a restriction enzyme site.
  • the competitor sequence was amplified using the same PCR primers as the target sequence. PCR products of the target sequence and the mutant sequence were identified by restriction analysis.
  • the amount of target sequence is the standard relative to the competing amplicon. It was estimated to be one copy of the competing sequence per total RNA at which the ratio of the target amplicon was 1: 1.
  • the primer sequences are as follows.
  • Antibodies to Mrtl were generated in rats.
  • a 15 amino acid peptide M15 (DGEGIHPSTPHRN GG) corresponding to positions 5-19 of Mrtl protein, and a Mrtla-specific carboxy terminus (positions 530-539) and an Mrtlb-specific carboxy terminus (positions 517-526), respectively
  • the corresponding peptides consisting of 10 amino acids, MIA-CIO (ARSQQRDVAT) and M1B-C10 (CELKWRKEEY) were synthesized (see Fig. 2C), cross-linked to keyhole limpet eraocyanin, and injected into rats.
  • an antiserum was prepared.
  • the obtained antiserum was purified on a peptide affinity column.
  • the peptide M1A-C12 (CELKWRKEN IFQ) corresponding to position 517-528 of Mrtla was used for the adsorption column.
  • Rat neocortical protein was prepared using 0.125% SDS, 0.625 sodium deoxycholate, 1.25% NP-40, 50 mM Tris-HCl (H 8.0), 150 mM NaCK ImM ED Extraction was performed using a buffer containing TA and Complete Mini (Roche).
  • Synaptosome and post-synaptic density (PSD) fractions were prepared from rat neocortex according to the method known in the literature (Carlin RK et al .; ⁇ Cell Biol, 1980, 86, 83, 843). Proteins were separated by 7.5% or 10% SDS-PAGE and transferred to Immun-Blot polyvinylidine difluoride membranes (Bio-Rad).
  • Antisense sequence targeting the sequence containing the start codon of rat mrtl gene (5'-cgtcctcgtccgc cttg-3 ', underlined is the nucleotide corresponding to the start codon) (hereinafter referred to as "AS2") or start A phosphorothioate oligodeoxynucleotide (hereinafter, referred to as "S-0 ⁇ ") having an antisense sequence (5'-ccccctacgcctccactcct-3 ') (hereinafter, referred to as "AS1”) targeting a sequence upstream of a codon is used. Changes in mRNA translated into brain Mrtl were examined in vivo.
  • S-0DN having a scrambled missense sequence corresponding to AS2 (5'-cgcctgttgtcgtcactccc-3 ') (hereinafter referred to as "Mis") or a solvent (phosphate-buffered saline: PBS) was used as a control. Injected. S-0DN was dissolved in PBS (2.5 g / L) and injected into the right ventricle of the rat by an osmotic minipump at an infusion rate of ⁇ iL / hour for 7 days.
  • PBS phosphate-buffered saline
  • the position of the tip of the injection needle inserted into the ventricle was determined using the stereotaxy device according to the rat brain diagram (Atlas of Paxinos and Watson), AP, -0.8 mm; V, +2.0; L, +1.5. (7) Statistical processing
  • RNA fingerprinting detected a 0.6 kb PCR product.
  • MAP administration 4.8 mg / kg
  • this PCR product increased in the neocortex of the mature rat but did not change in the neocortex of the young rat (see FIG. 1A).
  • FIG. 1A shows RNA fingers using arbitrarily primed PCR-printing from neocortex of mature rats (50 days old) and young rats (8 days old) treated with saline or MAP.
  • FIG. 14 is a diagram illustrating a result of detecting mrtl. As shown in Figure 1A, each group was amplified with increasing amounts of neocortical cDNA type I (2.5-IO I). In FIG. 1A, black arrows indicate the cDNA band containing the MAP and the A6021 sequence of mrtl (see FIG. 1B) that is differentially regulated by development.
  • Cloning and sequencing of the cDNA corresponding to the PCR product identified a novel rat gene, mrtl (MAP-responsive transcript 1), encoding a protein with PDZ-domain and PX-domain (SEQ ID NOS: 5 and 7). reference).
  • the putative protein which contains the gene F25H2.2, but is encoded by the gene F25H2.2 (43% homology with the Mrtl protein) lacks the glycine litz motif found at the amino terminus of MrU protein.
  • the mrtl transcript has two major splicing variants with different open reading frames (splicing variant ⁇ / ⁇ and variant III / IV), which are represented by two different variants in the Northern plot. (See Figures 1B and C).
  • FIG. 1B is a schematic diagram of the structure of mrtl cDNA.
  • white squares indicate the putative coding region, and bars indicate the sequence corresponding to the RAP-PCR clone A6021 and the hybridizing sequence. This shows the site recognized by the probe B41-3 and IVS.
  • FIG. 1C is a diagram showing the results of Northern plot of poly (A) + RNA (2 g / lane) derived from adult rat neocortex. As shown in Figure 1C, hybridization of the splicing variants III and IV with the selective probe to the IVS sequence, or hybridization of four splicing variants and the common probe to the B41-3 sequence with the common probe. Each identified one band (left lane) or two different bands (right lane).
  • the transcripts indicated by the lower band correspond to splicing variants I and II, and are presumed to encode a protein having a short hydrophilic carboxy terminus (Mrtl protein isoform, Mrtla), and the upper band
  • the transcripts indicated by (black arrows) correspond to splice variants III and IV and are presumed to encode a protein (MrU) 3 isoform (Mrtlb) lacking this structure.
  • SEQ ID NO: 5 shows the nucleotide sequence of mutant I of mrtl cDNA
  • SEQ ID NO: 6 shows the deduced amino acid sequence of Mrtla encoded by mutants I and II of mrtl cDNA.
  • mutant II of mrtl cDNA is represented by SEQ ID NO: 7, and mutants 111 and IV of mrtl cDNA are copied.
  • the deduced amino acid sequence of Mrtlb is shown in SEQ ID NO: 8.
  • FIG. 1D shows the Northern blot of poly (A) + RNA (lg / lane) derived from various brain sites and peripheral organs using a cDNA probe for IVS, B41-3 or j3-actin. It is a figure showing a result.
  • FIG. 2A N-terminal (5-19) amino acid residues of Mrtl (Ml-N15, see SEQ ID NOs: 6 and 8) common to Mrtla and MrUb, C-terminal specific to MrUa (530- 539)
  • Ml-N15 amino acid residues of Mrtl
  • Mrtlb amino acid residues of Mrtl
  • FIG. 2A is a schematic diagram showing the structure of Mrtla and Mrtlb and the recognition site of each antibody.
  • ⁇ -pan-Mrtl is a peptide M15 common to Mrtla and MrUb.
  • Q! -Mrtla and a-MrUb are the C-terminal peptides M1A-C10 specific to Mrtl and the C-terminal peptide M1B-specific to Mrtlb, respectively.
  • Figure 3 shows isoform-specific antibodies raised against C10.
  • G, PDZ and PX represent a glycin-rich domain, a PDZ domain and a PX domain, respectively, found in the Mrtl protein.
  • 2C is a diagram showing the amino acid sequences of the C-terminal of Mrtla and Mrtlb corresponding to the peptide used for preparing the isoform-specific anti-MrU antibody, and the underlined portion indicates the sequence common to Mrtla and Mrtlb. Show.
  • Mrtl protein was detected as a 62-kDa band by each antibody in western blots of neocortical extracts obtained from mature and young rats (FIG. 2B).
  • Figure 2B shows immature rats (8 days old) and
  • FIG. 4 shows the results of Western blot of a 25 zg protein sample extracted from the neocortex of an adult rat (50 days after birth). As indicated by the arrows, in these samples, the anti-pan_Mrtl antibody recognized the 62 kDa protein.
  • Mrtlb's M1B-C10 peptide contains the amino acid sequence found in Mrtla's MlA-C12 peptide, while the 62 kDa band recognized by the affinity-purified anti-Mrtlb antibody is in the presence of the M1B-C10 antigen. But disappeared in the presence of MIA-C12 antigen (FIG. 2D). These results indicate that the anti-Mrtlb antibody specifically recognizes Mrtlb protein (FIG. 2D).
  • FIG. 2D is a diagram showing the results of characterization of Mrtlb-specific antibodies.
  • FIG. 2E is a diagram showing the intracellular localization of Mrtl protein in neocortical tissue. As shown in Figure 2E, Mrtlb or pan-Mtl immunoreactivity was high for synaptosomes, but was detected for PSD isolated by a single treatment of synaptosomes with 0.5% Triton X-100. Was not done.
  • presynaptic marker proteins SNAP-25 and Kvl.4 were recognized by their antibodies in synaptosomes, but not in PSD. In contrast, Mrtla-specific immunoreactivity was not seen in synaptosomes and PSD fractions.
  • FIG. 3A is a schematic diagram of the recognition site of the mrtl mutant-specific primer and the putative PCR product.
  • the 5 'primer (white arrow) of the primer set for mrtla mRNA was designed to distinguish splicing boundaries (white arrow with *).
  • this set of primers amplifies a 126 bp mrtla mRNA-specific fragment (linear bar in the upper panel) without amplifying a 905 bp] nrtlb mRNA fragment (dotted bar in the lower panel).
  • FIG. 3B is a diagram showing PCR products amplified by a set of specific primers for mrtla and mrtlb in various samples.
  • the mrtla plasmid (lanes 1, 4 and 7), the mrtlb plasmid (lanes 2, 5 and 8) and the cDNA prepared from rat neocortical RNA (lanes 3, 6 and 9) were primers specific for mrtla. Amplification was performed with one set (group A or ⁇ ') or with a primer set specific for mrtlb (group B). Introducing a mutation (g to C) at one base adjacent to the 3 'end of the 5' primer for the mrtla mutant (group A ') eliminated the 905 bp nonspecific amplicon (see group A). ).
  • rRNA 28S ribosomal RNA
  • FIG. 3C is a diagram showing the results of co-amplification RT-PCR of a rat mrtlb-specific sequence and a 28S ribosomal RNA (rRNA) -specific sequence as an internal control.
  • rRNA ribosomal RNA
  • FIG. 3D is a diagram showing mrtla mRNA expression level and mrtlb mRNA expression level 1 hour after administration of acute MAP (4.8 mg / kg, sc). As shown in FIG. After one hour, mrtlb mRNA expression levels were selectively increased without affecting mrtla mRNA expression levels.
  • FIG. 4A is a diagram showing mrtlb mRNA expression levels at 1 hour, 3 hours, 6 hours, and 24 hours after a single MAP administration.
  • the data were obtained by competitive RT-PCR, and the control value was 8.0 ⁇ 1.3 amol / ⁇ total RNA (100%).
  • * indicates K0.0001 (compared to the control of saline treatment)
  • cross mark indicates K0.0001 (compared to 24 hours after MAP administration)
  • FIG. 4B shows the 8th day of birth
  • FIG. 4B shows the total mRNA expression levels from day 50 to day 50.
  • Mrtlb mRNA levels in the neocortex were increased by cocaine (30 mg / lg, sc), another behavioral sensitization-inducing dopamine agonist (see Figure 4C), but could be abused due to dependence-forming effects.
  • FIGS. 4C and 4D are diagrams showing the effects of acute administration of various drugs on the mrtlb mRNA expression level.
  • Sal represents saline
  • Coc represents cocaine
  • SCH represents SCH
  • Is SCH23390 and Pen is Pentobarbi.
  • ** indicates P * 0.01 (compared with saline-treated control, n 6), and in FIG.
  • MAP administration (4 mg / kg, ip, oligonucleotide injection) by injecting an antisense oligonucleotide complementary to the rat mrtl gene (including the putative translation start point of AS2 and mrtl) into the lateral ventricle for 7 consecutive days On the 3rd to 7th day).
  • an antisense oligonucleotide complementary to the rat mrtl gene including the putative translation start point of AS2 and mrtl
  • the injection of the antisense oligonucleotide did not affect the stereotype-inducing effect of MAP administration.
  • Antisense oligonucleotides (AS1) targeting a scrambled missense sequence (Mis), a solvent (PBS: phosphate buffered saline), or a sequence upstream of the initiation codon are associated with Mrtl expression and stereotypy induced by MAP administration. Action Failure to suppress behavioral augmentation indicates that the anti-mtll effect of AS2 is sequence-specific. Furthermore, unlike the Mrtl protein, injection of an antisense oligonucleotide (AS2) having an anti-mtll effect did not alter the basal and MAP-induced expression of the Arc protein in the striatum. It was shown that oligo nucleotides (AS2) having an anti- mr11 effect selectively act on the translation process of mrtl mRNA and do not act on the translation process of arc mRNA.
  • HMRTlcD 68 -87 (5'-acggctcgcctgctcgcaag-3 ') and the downstream primer hMRTlGl / 1202-1179 (5'-cacaggccgt tcac catccagagg-3') corresponding to the mrtlb-specific exon 12A sequence.
  • the temperature cycle is (1) 3 minutes at 94 ° C, (2) 2 minutes at 60 ° C, (3) 4 minutes at 72 ° C, (4) 45 seconds at 94 ° C, (5) 60 ° C 45 seconds, (6) 1 minute at 72 ° C, (7) 35 cycles of (4) to (5), (8) 5 minutes at 72 ° C.
  • the PCR product was cloned using the pGEM-T Easy TM Vector System, and the resulting 5 clones were analyzed using agarose gel and sequenced.
  • the translation initiation site of human mrtl was determined by the cap site hunting method using human brain cap site cDNA (NIPPON GENE). That is, the PCR amplification of the first round was performed using the rtl-oligo (IRC plus, 5 'gcgt tacaaggtacgccacagcg tatg-3') complementary to the sense DNA primer and the mrtl-specific corresponding to the first exon of human mrtl. Performed using antisense primer. A second nested PCR amplification was performed using r "oligo specific primers (2RC plus, 5'-gt tacaaggtacgccacagcgtatgatgc-3) and nrtl specific nested primers.
  • the PCR product was cloned into pGEM-T easy vector (Promega Japan K ..) and the resulting 6 clones were sequenced.
  • the transcription initiation site was determined by identifying a boundary sequence between the r-oligo and mrtl mRNA sequences.
  • the nucleotide sequence of the human mrtla cDNA sequenced as described above is shown in SEQ ID NO: 1, and the deduced amino acid sequence of human Mrtla encoded thereby is shown in SEQ ID NO: 2.
  • the nucleotide sequence of human mrtlb cDNA is shown in SEQ ID NO: 3, and the deduced amino acid sequence of human MrUb encoded thereby is shown in SEQ ID NO: 4.
  • a 261 bp 32 P-labeled probe corresponding to exon 2 and part of exons 1 and 3 of the sequence of human mrtl was prepared.
  • Northern Blot is Human Bra The test was performed using in Multiple Tissue Northern Blot II and Human Multiple Tissue Northern Blot (both from Clontech Laboratories, Inc.), and each lane contained 2 g of poly (A) + RNA. Hybridization to the probe was performed using Ultrahyb hybridization buffer according to the instructions (Ambion Inc.).
  • a schizophrenia-related protein a gene encoding the protein, a recombinant vector containing the gene, a transformant containing the recombinant vector, and an antibody against the protein or a fragment thereof.
  • a method for diagnosing schizophrenia or schizophrenia-like abnormalities and a diagnostic kit using the expression level in the brain of a gene encoding a schizophrenia-related protein as an index are provided.
  • a screening method and a screening kit for a substance for preventing and treating schizophrenia or a schizophrenia-like disorder using the effect of reducing the expression level of a gene encoding a schizophrenia-related protein as an index are provided.

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Abstract

L'invention concerne une protéine associée à la schizophrénie, un gène codant pour la protéine, un procédé de diagnostic de la schizophrénie ou d'une anomalie schizoïde en fonction du niveau d'expression intracérébrale du gène codant pour la protéine associée à la schizophrénie en tant qu'indicateur. L'invention porte également sur un kit de diagnostic, sur une méthode de criblage d'un agent préventif/thérapeutique pour la schizophrénie ou une anomalie schizoïde, en fonction de l'effet de réduction de l'expression du gène codant pour la protéine associée à la schizophrénie en tant qu'indicateur, et sur un kit de criblage. L'invention se rapporte notamment à une protéine associée à la schizophrénie, consistant en une séquence d'acides aminés SEQ ID N° 2, 4, 6 ou 8, ou en une séquence d'acides aminés SEQ ID N° 2, 4, 6 ou 8, dans laquelle au moins un acide aminé a été délété, substitué ou ajouté, le niveau d'expression intracérébrale fondamental étant réduit en suivant la croissance.
PCT/JP2004/006340 2003-09-30 2004-04-30 Proteine associee a la schizophrenie et gene codant pour cette derniere WO2005033308A1 (fr)

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CN110305949A (zh) * 2019-05-07 2019-10-08 中国人民解放军联勤保障部队第九0四医院 精神分裂症小鼠模型海马mRNA序列分析及试剂盒

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