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WO2002038763A1 - Gene pca2501 - Google Patents

Gene pca2501 Download PDF

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Publication number
WO2002038763A1
WO2002038763A1 PCT/JP2001/009545 JP0109545W WO0238763A1 WO 2002038763 A1 WO2002038763 A1 WO 2002038763A1 JP 0109545 W JP0109545 W JP 0109545W WO 0238763 A1 WO0238763 A1 WO 0238763A1
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WO
WIPO (PCT)
Prior art keywords
gene
pca2501
seq
protein
present
Prior art date
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PCT/JP2001/009545
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English (en)
Japanese (ja)
Inventor
Hideyuki Asaoka
Kenta Kaneda
Masakazu Adachi
Kazuo Miyanaga
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Japan Immunoresearch Laboratories Co Ltd
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Japan Immunoresearch Laboratories Co Ltd
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Priority to AU2002210985A priority Critical patent/AU2002210985A1/en
Priority to JP2002542078A priority patent/JP4112976B2/ja
Publication of WO2002038763A1 publication Critical patent/WO2002038763A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to a novel gene PCA2501 highly expressed in peripheral blood of schizophrenic patients. Further, the present invention provides a method for encoding a gene by the polynucleotide of the gene.
  • a polypeptide comprising an amino acid sequence to be expressed, and an expression product of the gene
  • the genetic information of an organism is accumulated as a sequence of four bases A, C, G, and T (DNA) present in the nucleus of a cell. This genetic information is used to maintain the lineage and ontogeny of each organism. Is stored in In humans, the number of bases of about 3 000 000 000 (3 X 1 0 9) We gutter, it is estimated that in it there are 5 to 00,000 genes.
  • This genetic information follows the flow in which mRNA is transcribed from a gene (DNA) and then translated into a protein, whereby regulatory proteins, structural proteins, or enzymes are made to maintain life phenomena. Abnormalities in the flow from the above genes to protein translation cause abnormalities in the life support system, such as cell proliferation and differentiation, and are thought to cause various diseases. From the results of the gene analysis to date, G protein-coupled receptors, insulin receptors, LDL receptors,
  • 51-1 Receptors such as HT receptor and dopamine receptor and related to the proliferation and differentiation of cells.Tyrosine kinase, histidine kinase, arginine kinase, protease, ATase, superoxide dismutase, histidine kinase
  • Tezzled-3 JP-A-11-253183
  • Frizzled- 4 2000-93186
  • genes related to nervous disorders such as schizophrenia and bipolar Z unipolar disorder.
  • a new human gene with the above-mentioned brain / nervous region, especially the region of schizophrenia can be provided, the gene expression level and its structure and function in each cell such as brain / nerve can be analyzed, and the expressed product It is thought that the analysis of the disease and the like will make it possible to elucidate, diagnose, and treat diseases related to the brain and nervous areas, particularly the schizophrenia area, for example, schizophrenia.
  • the aim is to provide human genes related to various schizophrenia. Disclosure of the invention
  • the present inventors have conducted intensive studies and, as a result, have analyzed the genes PCA2501 and PCA2501 polypeptide highly expressed in the blood of schizophrenic patients, and found that these genes are closely related to nervous system disorders, particularly schizophrenia.
  • the present inventors have found that they are useful for diagnosing these diseases and developing therapeutic agents, and have completed the present invention. That is, the present invention provides the following polynucleotide (a) or (b):
  • the present invention also relates to any one of the following (c), (d) or (e):
  • the present invention provides a gene consisting of
  • the present invention provides a gene expression product of the above gene, a recombinant expression vector having the above gene, a host cell having the expression vector, and a gene.
  • the present invention provides a polypeptide of the following (f) or (g): (f) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1,
  • the present invention further provides oligonucleotides, oligonucleotide probes and primers comprising at least 15 or 30 consecutive nucleotide sequences of the above-mentioned gene.
  • the present invention provides a method for detecting a PCA2501 gene or a gene expression product in a body fluid or a tissue sample, which binds to the oligonucleotide probe, using the above oligonucleotide probe. It provides a detection method.
  • the present invention provides a method for screening for a drug that interacts with the gene or gene expression product, characterized by using the above gene or gene expression product.
  • the present invention provides an antibody having a binding property to the gene expression product or a portion thereof.
  • the present invention provides a diagnostic agent for schizophrenia containing the above oligonucleotide probe or primer.
  • the present invention relates to a gene therapy agent containing an antisense strand oligonucleotide containing at least 15 or 30 contiguous nucleotide sequences of the PCA2501 gene as an active ingredient, and / or a PCA2501 activity inhibitor or an antipsychotic. It is intended to provide a disease agent.
  • the present invention provides a PCA2501 activity inhibitor or an antischizophrenic agent comprising an anti-PCA2501 antibody or a part thereof as an active ingredient. Further, it is a homologue of the gene of the present invention, which is a human, a dog, a monkey, a pig, a pig, and a human. And a homologue of a mammalian gene selected from the group of litter and cats. Furthermore, the present invention provides a screening method for identifying a compound that stimulates or suppresses the function of a PCA2501 polypeptide or a PCA2.501 gene expression product,
  • a screening method comprising a method selected from the group consisting of a method for detecting the effect of a candidate compound on the production of mRNA by encoding the polypeptide in a cell and the polypeptide.
  • the present invention also provides use of the above oligonucleotide probe or primer for the production of a diagnostic agent for schizophrenia.
  • the present invention provides a method for diagnosing schizophrenia, which comprises detecting the PCA2501 gene or gene expression product in a body fluid or tissue sample using the above oligonucleotide probe or primer. Is what you do. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a diagram showing a comparison of the expression level of the PCA2501 gene among healthy persons, schizophrenic patients, and other psychiatric patients.
  • FIG. 2 shows the relationship between the expression level of the PCA 2501 gene in schizophrenic patients and the psychiatric symptoms of the patients.
  • the PCA2501 protein encoded by the gene PCA2501 of the present invention is a FAS
  • the gene is not only a double-stranded DNA, but also a component thereof. It is intended to include each single-stranded DNA such as a sense strand and an antisense strand, and the length is not limited at all. Therefore, unless otherwise specified, the gene (DNA) of the present invention includes a double-stranded DNA containing human genomic DNA, a single-stranded DNA (sense strand) containing cDNA, and a sequence complementary to the sense strand. Single-stranded DNA (antisense strand), and fragments thereof. In the present invention, the gene (DNA) includes a leader sequence, a coding region, exons, and introns. Examples of the polynucleotide include RNA and DNA.
  • DNA includes cDNA, genomic DNA, and synthetic DNA.
  • a polypeptide having a specific amino acid sequence includes fragments, homologues, derivatives and variants thereof.
  • Variants are naturally occurring allelic variants, non-naturally occurring variants, deletions, substitutions, additions, and insertions; polynucleotide sequences that do not substantially alter the function of the encoded polypeptide. Means
  • amino acid sequence alterations may occur in nature, for example, due to mutations or post-translational modifications, etc., but may be artificially performed using naturally-occurring genes (for example, genes of the present invention). This can be done in a specific way.
  • the polypeptides include alleles, homologs, and naturally occurring variants that are at least 90%, preferably 95%, more preferably 98%, and even more preferably 99% homologous.
  • the gene expression product of the present invention has structural features that are commonly conserved, and the gene expression product of the present invention can be used for biological activity, for example, PCA2501 activity based on overexpression of the PCA2501 gene, or dopamine 1, dopamine 2, and noradrenaline in the brain. It has an overproduction activity of a sympathetic stimulant such as serotonin (5-HT;) and acetylcholine, a receptor stimulating activity of a sympathetic neurotransmitter receptor, and a transmission impairment activity of these neurotransmitters.
  • a sympathetic stimulant such as serotonin (5-HT;) and acetylcholine
  • the homology of polypeptides can be determined by measurement using sequence analysis software, for example, FASTA program (Clustal, V., Methods Mol. Biol., 25, 307-318 (1994)), or by SWISS-PROT. Can be analyzed.
  • Variant DNA means a silent or conservative variant of an amino acid substitution, Indicates a mutation in the base sequence in which the amino acid residue encoded by the base sequence does not change.
  • cysteine residues In addition to Valie or Leu, it is generally possible to delete or replace cysteine residues because one or more codons encoding a cysteine residue affect the disulfide binding of a particular polypeptide.
  • substitutions that are generally expected to produce the greatest change in protein properties are: a) a hydrophilic residue, for example, seryl or threonine, is substituted for a hydrophobic residue, for example, leucyl, isoleucyl, fanylalanil, 'valyl, or arayl;
  • Residues with very large side chains for example phenylalanine, can be replaced by those without side chains, such as glycine.
  • the provision of the gene PCA2501 of the present invention and its gene product provides extremely useful information and means for elucidation, understanding, diagnosis, prevention, and treatment of schizophrenia.
  • the gene of the present invention can also be suitably used in the development of a novel drug that suppresses the induction of the expression of the gene of the present invention, which is used for the treatment of schizophrenia.
  • detection of the expression of the gene of the present invention or expression of a product thereof in an individual or body fluid or tissue, and detection of mutation (deletion or point mutation) or abnormal expression of the gene can be performed by analyzing the above-mentioned schizophrenia, It can be suitably used in diagnosis.
  • the gene of the present invention specifically includes a gene comprising the nucleotide sequence of SEQ ID NO: 2 encoding the amino acid sequence represented by SEQ ID NO: 1, or a polynucleotide comprising the entire nucleotide sequence represented by SEQ ID NO: 2 or a complementary strand thereof
  • the gene is not particularly limited to these, and may be, for example, a gene encoding an amino acid sequence having a certain modification in the above specific amino acid sequence, or a gene encoding the above specific amino acid sequence or base sequence.
  • Can be a gene having the homology of The certain homology with the specific amino acid sequence or base sequence is, for example, at least 90% or more, preferably 9% or more.
  • It has a homology of 5% or more, more preferably 98% or more, and still more preferably 99% or more, and includes these homologs.
  • the amino acid sequence shown in SEQ ID NO: 1 A gene including a nucleotide sequence encoding an amino acid sequence in which one or more amino acids have been deleted, substituted or added (modified amino acid sequence) is also included.
  • the degree of “deletion, substitution or addition of amino acids” and their positions are determined by the fact that the modified protein has the same function as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 (PCA2501 protein). It is not particularly limited as long as it has the same effect as above, but preferably 1 to more than 10 and more preferably about 1 to several are preferably exemplified as having the same function as PCA2501 protein.
  • similar function means that the cause of schizophrenia is overproduction of sympathetic stimulants such as dopamine 1, dopamine 2, noradrenaline, serotonin (5-HT), and acetylcholine in the brain, and sympathetic neurotransmitters.
  • Abnormalities in the midbrain cortex and limbic system are caused by various causes such as receptor abnormalities and impaired transmission of these neurotransmitters, causing the appearance of mental illness and mental dysfunction. Since it is said that the hypothalamic-pituitary system causes an impairment of the motor function in the extracorporeal system and the hypothalamus-pituitary system, it can be said to be an activity similar to the activity of these PCA2501.
  • the PCA2501 activity includes, based on the overexpression of the PCA2501 gene, the overproduction activity of sympathetic stimulants such as dopamine 1, dopamine 2, noradrenaline, serotonin (5_HT) and acetylcholine in the brain, and the sympathetic neurotransmitter receptor.
  • sympathetic stimulants such as dopamine 1, dopamine 2, noradrenaline, serotonin (5_HT) and acetylcholine in the brain
  • the sympathetic neurotransmitter receptor examples include the activity of stimulating the receptor of the body and the activity of causing the impairment of the transmission of these neurotransmitters.
  • PCA2501 is a strong candidate for schizophrenia-related nervous system disorders. These properties are hereinafter referred to as “PCA2501 activity” or “PCA2501 polypeptide activity” or “biological activity of PCA2501”. Among these activities are also the antigenic and immunogenic activities of the PCA2501 polypeptide, especially the antigenic and immunogenic activities of the polypeptide of SEQ ID NO: 1.
  • the polypeptides of the present invention exhibit at least one biological activity of PCA2501.
  • the gene encoding the modified amino acid sequence is The PCA2501 gene of the present invention encoding the amino acid sequence before modification may be detectable.
  • examples of the DNA molecule of the present invention include a PCA2501 gene having a base sequence encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, but the PCA2501 gene is not particularly limited thereto. Genetic homologs are also included. .
  • the term "homolog of PCA2501 gene” means having homology to the PCA2501 gene of the present invention (or a gene product thereof), having the above-mentioned structural characteristics and commonality in gene expression patterns, and It refers to a series of related genes that are recognized as one gene family based on their similarity in biological function, and naturally includes alleles (alleles) of the PCA2501 gene.
  • the above-mentioned modification (mutation) of the amino acid sequence may occur in nature, for example, due to mutation or modification after translation.
  • a naturally derived gene for example, the human PCA2501 or human PCA2501 gene of the present invention
  • the present invention encompasses all modified genes having the above-mentioned properties, irrespective of the cause and means of such modification and mutation.
  • the gene (human PCA2501 gene) of the present invention also includes an allele (allele) of a gene encoding a protein having the amino acid sequence represented by SEQ ID NO: 1.
  • the above-mentioned artificial means include, for example, CytoSpecific Mutagenesis
  • DNA synthesis is performed by the phosphoramidite method or It can be carried out by a chemical synthesis by the triester method, or can be carried out on a commercially available automatic oligonucleotide synthesizer.
  • Double-stranded fragments are chemically synthesized by synthesizing a complementary strand and either annealing the strand together under appropriate conditions, or adding a complementary strand using a DNA polymerase with the appropriate primer sequence. It can also be obtained from single stranded products.
  • a gene having the base sequence shown in SEQ ID NO: 2 can be exemplified.
  • the coding region in this nucleotide sequence shows one combination example of codons indicating each amino acid residue in the amino acid sequence shown in SEQ ID NO: 1.
  • the gene of the present invention is not limited to a gene having such a specific base sequence, but may have a base sequence selected by combining arbitrary codons with each amino acid residue. Codons can be selected according to a conventional method, for example, considering the frequency of codon usage of the host to be used, CNcleic Acids Res., 9, 43 (1981)].
  • the DNA molecule of the present invention also includes a DNA molecule having a certain homology with the base sequence shown in SEQ ID NO: 2.
  • the above-mentioned homology refers to a polynucleotide having at least 70% identity, preferably at least 90% identity, more preferably at least 95% identity with the nucleotide sequence shown in SEQ ID NO: 2 and a polynucleotide complementary thereto. To tell.
  • Such genes include, for example, those set forth in SEQ ID NO: 2 under stringent conditions of 50 ° C in 0.2XS SC containing 0.1% SDS or 60 ° C in 1XS SC containing 0.1% SDS.
  • a gene having a base sequence that hybridizes with DNA consisting of a base sequence to be used can also be exemplified.
  • the gene of the present invention can be easily produced and obtained by general genetic engineering techniques on the basis of sequence information on specific examples of the gene of the present invention disclosed by the present invention [Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); See Sequential Chemistry Laboratory Course “Gene Research Methods I, II and III", edited by The Biochemical Society of Japan (1986)]. Specifically, from an appropriate source for expressing the gene of the present invention, c A DNA library can be prepared and the desired clone can be selected from the library using an appropriate probe or antibody specific to the gene of the present invention (Proc. Natl. Acad. Sc USA., 78, 6613 ( 1981); Science, 222, 778 (1983) and the like].
  • examples of the origin of cDNA include various cells and tissues expressing the gene of the present invention, and cultured cells derived therefrom. Separation of total RNA therefrom, isolation and purification of mRNA, acquisition of cDNA, and cloning thereof can all be carried out in accordance with conventional methods.
  • cDNA libraries are commercially available. In the present invention, cDNA libraries such as various cDNA libraries commercially available from Clontech Lab. Inc. and the like are used. Can also.
  • the method for screening the gene of the present invention from a cDNA library is not particularly limited, either, and ordinary methods can be followed. Specifically, for example, for a protein produced by cDNA, a method of selecting a corresponding cDNA clone by immunoscreening using a specific antibody of the protein, a method of selectively selecting a target DNA sequence Examples include plaque hybridization, colony hybridization using a probe to be bound, and a combination thereof.
  • DNA or the like chemically synthesized based on the information on the nucleotide sequence of the gene of the present invention can be generally used, but the gene of the present invention or a fragment thereof which has already been obtained can also be used favorably. it can.
  • sense primers and antisense primers set based on the nucleotide sequence information of the gene of the present invention can also be used as screening probes.
  • the nucleotide sequence used as the probe is a partial nucleotide sequence corresponding to SEQ ID NO: 2, and has at least 15 consecutive bases, preferably 2
  • a probe having 0 consecutive bases is also included, or a positive clone having the above sequence itself can be used as a probe.
  • a DNAZRNA amplification method by a PCR method [Science, 230, 1350 (1985)] can be suitably used.
  • the RACE method Rapid amplification of cDNA ends; Experimental Medicine, 12 (6), 35 (1994)
  • the 5'-RACE method [ Natl. Acad. Sci., USA., 8, 8998 (1988)].
  • Primers to be used when employing such a PCR method can be appropriately set based on the sequence information of the gene of the present invention revealed by the present invention, and can be synthesized according to a conventional method.
  • isolation and purification of the amplified DNAZRNA fragment can be performed by a conventional method as described above, for example, by gel electrophoresis.
  • the gene or various DNA fragments of the present invention obtained as described above can be prepared by a conventional method, for example, the dideoxy method OProc. Natl. Acad. Sci., USA., 74, 5463 (1977)] or the Maxam-Gilbert method CMethods in Enzymology, 65, 499 (1980)], or simply using a commercially available sequence kit or the like.
  • the presence or absence of the expression of the gene of the present invention in an individual or various tissues can be specifically detected by utilizing a part or all of the nucleotide sequence of the gene. be able to.
  • Such detection can be performed by a conventional method, for example, RT-PCR [Reverse transcribed-Polymerase chain reaction; E. S. Kawasaki, et al.,
  • RNA-level measurement using in situ hybridization etc.
  • NASBA method Nucleic acid sequence-based amplification, Nature, 350, 91-92 (1991) 3 and various other methods.
  • a detection method by RT-PCR can be mentioned.
  • primers used in the case of employing the PCR method are not particularly limited as long as they are specific to the gene of the present invention and can specifically amplify only the gene of the present invention. And can be set appropriately.
  • primers include those having a partial sequence of the gene of the present invention having a length of about 10 to 35 nucleotides, preferably about 15 to 30 nucleotides.
  • the gene of the present invention also includes a DNA fragment used as a specific primer and / or a specific probe for detecting the PCA2501 gene according to the present invention.
  • the DNA fragment can be defined as DNA characterized in that it hybridizes under stringent conditions to DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2.
  • stringent conditions include ordinary conditions used as a primer or a probe, and are not particularly limited, and include, for example, 0.2% SDS containing 0.1% SDS as described above.
  • An example is a condition of 50 ° C or a condition of 60 ° C in 33 ° per one hour containing 0.1% 303.
  • the gene product (PCA2501 protein) or a protein containing the gene product can be easily and stably produced in a large amount by using ordinary genetic engineering techniques.
  • the present invention relates to a protein such as a PCA2501 protein encoded by the gene of the present invention, a vector for producing the protein, for example, a vector containing the gene of the present invention, a host cell transformed with the vector,
  • the present invention also provides a method for producing the protein of the present invention by culturing the host cell.
  • a specific embodiment of the protein of the present invention has the amino acid sequence shown in SEQ ID NO: 1.
  • PCA2501 protein may be mentioned, and the protein of the present invention includes the PCA2
  • Such homologues include In the amino acid sequence shown in SEQ ID NO: 1, there may be mentioned a protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added, and having the above-mentioned activity of PCA2501.
  • the PCA2501 activity refers to dopamine 1, dopamine 2, noradrenaline, and seroto in the brain based on the overexpression of the PCA2501 gene.
  • Excessive activity of sympathetic stimulants such as acetylcholine (5-HT), acetylcholine, etc., receptor stimulating activity of sympathetic neurotransmitter receptors, and activity of these neurotransmitters to cause impaired transmission can be exemplified.
  • Specific examples include gene products of homologues of the PCA2501 gene (PCA2501-equivalent genes including alleles).
  • homologs of the PCA2501 protein of the present invention include mammals, such as humans, pests, higgies, pests, dogs, monkeys, which have the same activity as the PCA2501 protein having the amino acid sequence shown in SEQ ID NO: 1. It also includes rodent proteins such as cats, bears, rats, and egrets.
  • the protein of the present invention can be obtained by a conventional gene recombination technique [for example, Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., Based on the PCA2501 gene sequence information provided by the present invention. 130, 692 (1985); Proc. Natl. Acad. Sci., USA., 80, 5990 (1983), etc.].
  • the production of the protein is carried out by preparing a recombinant DNA (expression vector) capable of expressing the gene encoding the desired protein in a host cell, introducing this into a host cell, and transforming the host. This is carried out by culturing the transformant and then recovering the resulting culture.
  • a recombinant DNA expression vector
  • prokaryote and eukaryote can be used as the host cell.
  • prokaryote hosts include a wide range of commonly used hosts such as Escherichia coli and Bacillus subtilis. Escherichia Kori among others
  • eukaryotic host cells include cells of vertebrates, yeast, and the like. COS cells [Cell, 23: 175 (1981)] and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains [Pro Natl. Acad. Sci., USA., 77: 4216 (1980) As the latter, Saccharomyces yeast cells and the like are preferably used. Of course, it is not limited to these.
  • a promoter capable of expressing the gene of the present invention in this vector using a vector capable of replicating in the host cell is used to promote the promoter and the SD (Shine).
  • SD Secure Digital
  • a plasmid derived from Escherichia coli, for example, pBR322, pBR325, pUC12, pUC13, etc. is often used.
  • the present invention is not limited thereto, and various known vectors can be used.
  • Examples of commercial products of the above vector used in an expression system using Escherichia coli include, for example, pGEX-4T (Amers am Pharmacia Biotech II) pMAL-C2, pMA1-P2 (New England Biolabs) , PET21, pET21 / 1 acq (Invitrogen), pBAD / His (Invitrogen) and the like.
  • the expression vector When a vertebrate cell is used as a host, the expression vector usually includes a promoter, an RNA splice site, a polyadenylation site, and a transcription termination sequence located upstream of the gene of the present invention to be expressed. It may also have a replication origin if desired. Specific examples of the expression vector include, for example, pSV2dhfr (Mol.Cell.
  • pAc 5 / V5-H is, pMT / V 5 -H is, pMT And insect cell vectors such as / Bip / V 5-his (all from Invitrogen).
  • a specific example of an expression vector when a yeast cell is used as a host is, for example, PAM82 having a promoter for an acid phosphatase gene (Proc. Natl. Acad. Sci., USA., 80: 1 (1983) And the like.
  • Commercially available expression vectors for yeast cells include, for example, pPICZ (Invitrogen) and pPICZ (Invitrogen).
  • the promoter No particular limitation is imposed on the promoter.
  • Escherichia genus for example, tryptophan (trp) promoter, ⁇ ⁇ promoter, lac promoter, recA promoter, PLZPR promoter and the like can be preferably used.
  • the host is Bacillus, SP 0 1 promoter one, SP 0 2 flops port motor - and p en p promoter are preferred.
  • yeast is used as a host, for example, a pHO5 promoter, a pgk promoter, a GAP promoter, an ADH promoter and the like can be suitably used.
  • preferred promoters include SV40-derived promoters, retrovirus promoters, metamouth thionein promoters, heat shock promoters, cytomegalovirus promoters, and SR promoters.
  • the CK promoter can be exemplified.
  • a normal fusion protein expression vector can also be preferably used.
  • the vector include pGEX (Promega) for expression as a fusion protein with dalhithion-S-transferase'ase (GST).
  • examples of the polynucleotide sequence whose coding sequence of the mature polypeptide assists the expression and secretion of the polypeptide from a host cell include a secretory sequence and a leader sequence, and are used for purification of a fusion mature polypeptide from a bacterial host.
  • Marker sequence hexahistidine 'tag, histidine' tag
  • HA hemagglutinin
  • a method for introducing a desired recombinant DNA (expression vector) into a host cell and a method for transforming the same are not particularly limited, and various general methods can be employed.
  • the resulting transformant can be cultured according to a conventional method, and the target protein of the present invention encoded by a gene designed as desired by the culture is expressed in the transformant, outside the cell or on the cell membrane of the transformant. Produced (accumulated, secreted).
  • the medium used for the cultivation various types of media commonly used depending on the host cells used can be appropriately selected and used, and the cultivation can be carried out under conditions suitable for the growth of the host cells.
  • the recombinant protein of the present invention thus obtained can be subjected to various separation operations utilizing its physical properties, chemical properties, and the like [“Biochemical Data Book II”, pp. 1175-1259, 1st edition, 1st edition, if desired. Press, June 23, 1980, issued by Tokyo Chemical Dojin, Ltd .; see Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987), etc.). .
  • the method include ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, ultrasonic crushing, ultrafiltration, molecular sieve chromatography (gel filtration). , Adsorption chromatography, ion-symmetry chromatography, affinity chromatography, various liquid chromatography such as high performance liquid chromatography (HPLC), dialysis method, and combinations thereof. Affinity chromatography using a column to which a specific antibody against a protein is bound can be exemplified.
  • the nucleotide sequence of the PCA2501 gene shown in SEQ ID NO: 2 can be used favorably.
  • a codon indicating each amino acid residue can be appropriately selected and changed as needed for use.
  • amino acid sequence encoded by the PCA2501 gene When the amino acid residue or amino acid sequence is modified by substitution, deletion, addition or the like, it can be carried out by the above-mentioned various methods such as cytosolic mugenesis.
  • the protein of the present invention can be produced by a general chemical synthesis method according to the amino acid sequence shown in SEQ ID NO: 1.
  • the method includes a method for synthesizing a peptide by a usual liquid phase method and a solid phase method.
  • the peptide synthesis method includes a so-called stepwise gelation method in which amino acids are sequentially linked one by one based on amino acid sequence information to extend the chain, and a fragment comprising several amino acids. And a fragment condensation method in which each fragment is then subjected to a coupling reaction.
  • the synthesis of the protein of the present invention may be performed by any of these methods.
  • the condensation method employed in the above peptide synthesis can also be in accordance with a conventional method, for example, an azide method, a mixed acid anhydride method, a DCC method, an active ester method, a redox method, a DPPA (diphenyl phosphoryl azide) method, DCC + additive (1-hydroxybenzotriazole, N-hydroxysuccinamide, N-hydroxy-15-norpolneno, -2,3-dicarpoxyimide, etc.) method, Woodward method and the like can be exemplified.
  • the solvent that can be used in each of these methods can also be appropriately selected from well-known general ones used in this kind of peptide condensation reaction.
  • Examples thereof include dimethylformamide (DMF;), dimethylsulfoxide (DMS ⁇ ), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and a mixed solvent thereof.
  • DMF dimethylformamide
  • DMS ⁇ dimethylsulfoxide
  • THF tetrahydrofuran
  • ethyl acetate examples thereof include dimethylformamide (DMF;), dimethylsulfoxide (DMS ⁇ ), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and a mixed solvent thereof.
  • the hepoxyl group in the amino acid or peptide not involved in the reaction is generally converted into a lower alkyl ester such as a methyl ester, an ethyl ester or a tertiary butyl ester by esterification, for example, a benzyl ester, ⁇ - It can be protected as aralkyl esters such as methoxybenzyl ester and p-nitrobenzyl ester.
  • amino acid having a functional group in the side chain for example, the hydroxyl group of a tyrosine residue may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, etc., but such protection is not always required. No need to do.
  • the guanidino group of an arginine residue includes a nitro group, a tosyl group, a p-methoxybenzenesulfonyl group, a methylene-12-sulfonyl group, a benzyloxycarbonyl group, an isoporyloxycarbonyl group, and an adamantyl. It can be protected by a suitable protecting group such as an oxycarbonyl group.
  • the deprotection reaction of these protecting groups in the amino acids and peptides having the above protecting groups and the finally obtained protein of the present invention can also be carried out by commonly used methods such as catalytic reduction method, liquid ammonia / sodium, hydrogen fluoride, bromide, etc. It can be carried out according to a method using hydrogen, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid and the like.
  • the protein of the present invention thus obtained is appropriately purified according to the above-mentioned various methods, for example, a method widely used in the field of peptide chemistry such as ion-exchange resin, partition chromatography, gel chromatography, and countercurrent partition method. It can be carried out.
  • a method widely used in the field of peptide chemistry such as ion-exchange resin, partition chromatography, gel chromatography, and countercurrent partition method. It can be carried out.
  • the PCA2501 protein of the present invention can be suitably used as an immunizing antigen for producing a specific antibody thereof.
  • a desired antiserum (polyclonal antibody) and monoclonal antibody can be used.
  • An antibody can be obtained.
  • the antibody thus obtained can be advantageously used, for example, for purification of the PCA2501 protein and for its determination by immunological techniques without discrimination. More specifically, since amplification and enhanced expression of the gene of the present invention have been confirmed in a blood sample of a schizophrenic patient, the antibody can be used to diagnose schizophrenia or schizophrenia. Can be used to determine the degree of progress.
  • the PCA2501 antibody of the present invention obtained as described above is useful in the pharmaceutical field as a pharmaceutical containing the PCA2501 antibody as an active ingredient. Therefore, the present invention also provides a pharmaceutical composition comprising the PCA2501 antibody of the present invention as an active ingredient.
  • the usefulness of the PCA2501 antibody of the present invention as a medicine is, as described above, an overproduction effect of a sympathetic stimulant possessed by a specific polypeptide of a schizophrenic patient, and a receptor of a sympathetic neurotransmitter receptor.
  • Examples of the action include an abnormal body stimulating action and an active action of suppressing or reducing a disorder caused by a neurotransmitter transmission disorder. Confirmation of these antibody activities can be confirmed by a usual immunological antigen-antibody reaction test.
  • the protein as an active ingredient in the pharmaceutical composition also includes a pharmaceutically acceptable salt thereof.
  • Such salts include non-toxic alkali metal salts such as sodium, potassium, lithium, calcium, magnesium, barium, ammonium, alkaline earth metal salts, ammonium salts, etc., prepared by methods well known in the art. Included. Further, the above salts also include non-toxic acid addition salts obtained by reacting the protein of the present invention with an appropriate organic or inorganic acid.
  • Non-toxic acid addition salts include, for example, hydrochloride, hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, Borate, benzoate, lactate, phosphate, p-toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate, tartrate, sulfonate, glycolic acid Salts, maleates, ascorbates, benzenesulfonates and napsylates are exemplified.
  • compositions include those containing the protein of the present invention as an active ingredient and a pharmaceutically effective amount thereof together with a suitable non-toxic pharmaceutical carrier or diluent.
  • Pharmaceutical carriers that can be used in the above pharmaceutical composition include fillers, bulking agents, binders, humectants, disintegrants, surfactants, and lubricants that are usually used depending on the use form of the formulation
  • diluents or excipients such as a preparation can be exemplified. It is appropriately selected and used depending on the given unit form.
  • Particularly preferred pharmaceutical preparations of the present invention include various components which can be used in ordinary protein preparations, such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH regulators, surfactants It is prepared by using such as appropriate.
  • the stabilizer examples include human serum albumin, ordinary L-amino acids, saccharides, and cellulose derivatives. These can be used alone or in combination with a surfactant. In particular, according to this combination, there are cases where the stability of the active ingredient can be further improved.
  • the L-amino acid is not particularly limited, and may be, for example, any of glycine, cysteine, and glumic acid.
  • sugars there is no particular limitation on the above-mentioned sugars, and for example, monosaccharides such as glucose, mannose, galactose and fructose, sugar alcohols such as mannitol, inositol and xylitol, disaccharides such as sucrose, maltose and lactose, dextran, hydroxypropyl starch And polysaccharides such as chondroitin sulfate and hyaluronic acid, and derivatives thereof.
  • monosaccharides such as glucose, mannose, galactose and fructose
  • sugar alcohols such as mannitol, inositol and xylitol
  • disaccharides such as sucrose, maltose and lactose
  • dextran hydroxypropyl starch
  • polysaccharides such as chondroitin sulfate and hyaluronic acid, and derivatives thereof.
  • the surfactant is not particularly limited, and both ionic and nonionic surfactants can be used.
  • ionic and nonionic surfactants can be used.
  • polyoxyethylene glycol sorbitan alkyl ester polyoxyethylene alkyl ether, sorbitan monoester Or fatty acid glyceride.
  • cellulose derivative there is no particular limitation on the cellulose derivative, and methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and the like can be used.
  • the amount of the saccharide to be added is about 0.001 mg or more per l ⁇ g of the active ingredient, and preferably about 0.01 to 1 Omg.
  • the amount of the surfactant added is about 0.001 mg or more, preferably about 0.01 mg, per 1 g of the active ingredient. It is appropriate that the range is about 0001 to 0.0 lmg.
  • the amount of human serum albumin to be added is about 0.001 mg or more, preferably about 0.001 to 0.1 mg per tg of the active ingredient.
  • the amino acid is suitably used in an amount of about 0.001 to 1 Omg per 18 active ingredients.
  • the amount of the cellulose derivative to be added is suitably about 0.000 lmg or more, preferably about 0.001 to 0.1 mg per 1 g of the active ingredient.
  • the amount of the active ingredient contained in the pharmaceutical preparation of the present invention is appropriately selected from a wide range, but is usually about 0.0001 to 70% by weight, preferably about 0.0001 to 5% by weight. is there.
  • various additives such as a buffer, an isotonic agent, a chelating agent and the like can be added to the pharmaceutical preparation of the present invention.
  • a buffer boric acid, phosphoric acid, acetic acid, citric acid, ⁇ -aminocaproic acid, glutamic acid and / or a salt thereof (for example, sodium salt, potassium salt, calcium salt, magnesium salt) Alkali metal salts such as salts and alkaline earth metal salts).
  • the isotonic agent include sodium chloride, potassium chloride, saccharides, glycerin and the like.
  • the chelating agent include sodium edetate and citric acid.
  • the pharmaceutical preparation of the present invention can be used not only as a solution preparation, but also by lyophilizing it to make it storable, dissolving it in a buffer solution containing water for use, a saline solution, or the like, at an appropriate concentration. It is also possible to use it after preparing it.
  • various forms can be selected according to the purpose of treatment, and typical examples are tablets, pills, powders, powders, granules, capsules and the like.
  • Solid dosage forms, and liquid dosage forms such as solutions, suspensions, emulsions, syrups, and elixirs, depending on the route of administration. These also include oral, parenteral, nasal, vaginal, and suppository forms.
  • Preparations, sublingual preparations, ointments and the like and can be prepared, molded or prepared according to the usual methods.
  • the above-mentioned preparation carrier may be, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, caiiic acid, potassium phosphate, etc., water, etc.
  • Binders such as ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, propyloxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone, sodium carboxymethylcellulose, carboxymethylcellulose calcium, low substitution degree Disintegrators such as hydroxypropylcellulose, dried starch, sodium alginate, powdered agar, powdered laminar, sodium hydrogencarbonate, calcium carbonate, polyoxyethylene sorbitan fat Surfactants such as acid esters, sodium lauryl sulfate, and monoglyceride stearate; crushing inhibitors such as sucrose, stearin, cocoa batata, and hydrogenated oil; quaternary ammonium bases; and absorption promoters such as quaternary ammonium base and sodium lauryl sulfate Uses humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite
  • the tablets can be tablets coated with a usual coating as required, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, and double tablets or multilayer tablets. it can.
  • excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, etc.
  • binders such as gum arabic powder, tragacanth powder, gelatin, ethanol, etc. , Laminaran, agar and other disintegrating agents can be used.
  • Capsules are usually prepared by mixing the active ingredient of the present invention with the various pharmaceutical carriers exemplified above and filling the mixture into hard gelatin capsules, soft capsules and the like according to a conventional method.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable solutions including conventional inert diluents such as water, emulsions, suspensions, syrups, elixirs and the like.
  • auxiliaries such as wetting agents, emulsions, and suspending agents can be added, and these are prepared according to a conventional method.
  • liquid dosage forms for parenteral administration such as sterile aqueous to non-aqueous solutions, emulsions and suspensions
  • diluents such as water, ethyl alcohol, propylene glycol, polyethylene glycol, ethoxylated isostearyl
  • Vegetable oils such as alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester and olive oil can be used, and injectable organic esters such as ethyl oleate can be blended.
  • injectable organic esters such as ethyl oleate can be blended.
  • These may further contain conventional solubilizers, buffers, wetting agents, emulsifiers, suspending agents, preservatives, dispersants, and the like.
  • Sterilization can be performed by, for example, a filtration operation of passing through a bacteria-retaining filter, combination of a bactericide, irradiation treatment, and heat treatment. They can also be prepared in the form of sterile solid compositions which can be dissolved in sterile water or a suitable sterilizable medium immediately before use.
  • preparation carriers such as polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin and semisynthetic dalyceride can be used.
  • diluents such as white petrolatum, paraffin, glycerin, cellulose derivatives, propylene glycol, polyethylene glycol, silicon, bentonite and oil of olive oil Vegetable oils and the like can be used.
  • composition for nasal or sublingual administration can be prepared according to a conventional method using well-known standard excipients.
  • the drug of the present invention may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals, if necessary.
  • the amount of the active ingredient to be contained in the above pharmaceutical preparation and the dose thereof are not particularly limited, and may vary widely depending on the desired therapeutic effect, administration method, treatment period, patient age, gender and other conditions. It is appropriately selected. In general, the dose is usually about 0.01 g to 10 mg, preferably about 0.1 ng to 1 mg per kg of body weight per day. Can be administered in one or several divided doses per day.
  • the gene of the present invention is highly expressed in the peripheral blood of schizophrenic patients, so that the whole or part of the antisense strand of the PCA2501 gene of the present invention is expressed.
  • dopamine 2 and dopamine 2 in the brain based on overexpression of the PCA2501 gene causes excessive production of sympathetic stimulants such as noradrenaline, serotonin (5-HT), and acetylcholine, causes stimulatory activity of sympathetic neurotransmitter receptors, or causes impairment of transmission of these neurotransmitters Suppresses activity and consequently improves functional findings such as neuropsychiatric symptoms, motor function, endocrine function, etc. in schizophrenic patients
  • gene therapy composition having ability antipsychotic activity or anti-P C A 2 5 0 1 activity, or considered to be utilized as a gene therapy agent.
  • the present invention provides a gene therapy vector containing the whole or part of the antisense strand of the PCA2501 gene, and a cell into which the antisense strand of the PCA2501 gene has been introduced using the vector as an active ingredient. To provide medical supplies It is.
  • the vector for gene therapy introduction comprising the antisense strand of the PCA2501 gene containing all or a part of the nucleotide sequence represented by SEQ ID NO: 2 and the PCA2501 gene antisense strand And a gene therapy agent comprising, as an active ingredient, a cell into which PCA2501 gene antisense strand has been introduced, and a cell into which PCA2501 gene antisense strand has been introduced by the vector and the vector for gene therapy.
  • the PCA2501 gene containing the antisense strand of the PCA2501 gene containing the whole or part of the nucleotide sequence of SEQ ID NO: 2 By administering cells into which antisense chains have been introduced into the brain of a schizophrenic patient or to the vascular tissue site of the patient, the overactive activity of sympathetic stimulants in these tissues, the receptor for the sympathetic neurotransmitter receptor A treatment for schizophrenia characterized by suppressing the stimulating activity or the activity of impairing the transmission of these neurotransmitters or suppressing the expression level of the PCA2501 gene in these cells, and a PCA2501 activity inhibitor or antipsychotic drug Can be provided.
  • a medicine containing, as an active ingredient, a viral vector for gene therapy introduction containing the PCA2501 gene antisense chain, in particular, an effect of overproducing PCA2501 activity or a sympathomimetic substance.
  • the present invention also provides a medicament for use in a treatment for suppressing a receptor stimulating activity of a sympathetic neurotransmitter receptor or a transmission impairment activity of these neurotransmitters.
  • the present invention relates to a cell expressing the PCA2501 gene of the present invention, which has a sequence complementary to intracellular mRNA: an antisense drug for producing RNA, inhibiting translation and suppressing PCA2501 gene expression
  • an antisense drug for producing RNA inhibiting translation and suppressing PCA2501 gene expression
  • the therapy may involve inhibiting the transcription or translation process, for example, by binding to the native mRNA of the PCA2501-expressing cell carrying the PCA2501 gene, or by intercalating between the DNA duplexes to form a triplex.
  • This is a method for suppressing the expression of a target gene.
  • it can be considered as a method for producing an antisense oligonucleotide complementary to the mRNA of the gene and supplying the antisense oligonucleotide to target cells.
  • the PCA2501 activity in the recipient cell / target cell can be suppressed.
  • the vector can be maintained outside the chromosome using a vector or a plasmid containing the antisense oligonucleotide, and introduced into a target cell.
  • a desired inhibitory effect can be obtained by incorporating a sense oligonucleotide and infecting the cells expressing PCA2501 activity with this to overexpress the antisense oligonucleotide.
  • the antisense oligonucleotide When the antisense oligonucleotide is introduced into cells having the PCA2501 gene to suppress the expression of the PCA2501 protein, the antisense oligonucleotide does not need to correspond to the full length of the corresponding PCA2501 gene.
  • the PCA2501 gene retains a function that is substantially the same as the function of suppressing the expression function of the PCA2501 gene, even if it is the above-described variant, it also uses a gene consisting of a partial sequence that retains a specific function. You can also.
  • Vectors for the introduction of the desired gene for both recombination and extrachromosomal maintenance are already known in the art, and any of the known vectors can be used in the present invention.
  • a viral vector or a plasmid vector containing a copy of the antisense oligonucleotide of PCA2501 linked to an expression control element and capable of expressing the antisense oligonucleotide product in a target cell can be mentioned.
  • the above-mentioned expression vector can be usually used, but preferably, for example, as an origin vector, US Pat. No.
  • a promoter used in a vector used in gene transfer therapy a promoter specific to an affected tissue to be treated for various diseases can be suitably used.
  • liver albumin, -fetoprotein, 1-antitrypsin, transferrin, transstyrene, etc.
  • examples thereof include carboxylate anhydrase I and carcinoenprogen antigen.
  • estrogen, aromatase cytochrome p450, cholesterol side chain cleavage P450, 17-alpha hydroxylase P450 and the like can be exemplified.
  • prostate antigen for the prostate, prostate antigen, gp91-fox gene, prostate-specific kallikrein and the like can be exemplified.
  • breast erb-B2, erb_B3, / 3-casein, 3-lactoglobin, whey protein and the like can be exemplified.
  • the activator protein C-globulin can be exemplified.
  • skin examples include K-141-keratin, human keratin 1 or 6, and leucrin.
  • examples include glial fibrillary acidic protein, mature astrocyte specific protein, myelin basic protein, tyrosine hydroxylase, and the like.
  • examples include villin, glucagon, and amyloid polypeptide of the islet of Langerhans.
  • examples include villin, glucagon, and amyloid polypeptide of the islet of Langerhans.
  • thyroglobulin, calcitonin and the like can be exemplified.
  • ⁇ -collagen, osteocalcin, bone sialoglycoprotein and the like can be exemplified.
  • renin, liver, bone, kidney alkaline phosphatase, erythropoietin, etc. and for the kidney, amylase, PAP1, etc. can be exemplified.
  • the antisense oligonucleotide to be introduced (all or a part of the complementary sequence corresponding to the PCA2501 gene sequence) is determined based on the base sequence information of the PCA2501 gene of the present invention. As described above, it can be easily produced and obtained by general genetic engineering techniques.
  • PCA2501 anti-sense- The cells transformed with the oligonucleotide can be used as a drug for suppressing PCA2501 activity in a state of isolation in itself, or as a model system for therapeutic research.
  • the antisense oligonucleotide introduction vector described above can be introduced into a patient's target cells by local or systemic injection into the target tissue site of the patient. it can. At this time, by systemic administration, it is possible to reach any cells that can express PCA250mRNA at other sites. If the transduced gene is not permanently incorporated into the chromosome of each target cell, this can be achieved by repeating the administration periodically.
  • the gene therapy method of the present invention includes an in vivo method in which the material for introducing an antisense oligonucleotide (the vector for introducing an antisense oligonucleotide) described above is directly injected into the body. It includes both the ex vivo method, in which the target cell is once removed, the gene is introduced outside the body, and the cell is then returned to the body.
  • lipozyme which is an active molecule that cleaves an RNA chain by introducing an antisense oligonucleotide directly into PCA2501 into a cell, is also possible.
  • a vector for gene transfer containing all or a fragment of an antisense oligonucleotide of a sequence corresponding to PCA2501 of the present invention, which will be described later, and a human PCA2501 being transformed by the vector into an antisense oligonucleotide.
  • the gene therapy agent of the present invention containing the nucleotide-introduced cell as an active ingredient is particularly intended for schizophrenic patients, but the above-described gene therapy (treatment) is not limited to schizophrenic patients. Can also be used for the treatment of schizophrenia complications, as well as for gene labeling.
  • a target cell In addition to brain and nervous tissue, brain cells, brain nerve cells, and cells of tissues where PCA2501 expression is observed, heart, placenta, lung, liver, spleen, spleen, small intestine, peripheral blood tissues, nerve cells, lymphocytes , Fibroblasts, hepatocytes, hematopoietic stem cells, and the like.
  • the method of introducing antisense oligonucleotides in the above gene therapy includes a viral introduction method and a non-viral introduction method.
  • An example of a viral introduction method includes a method using a retrovirus vector as a vector in consideration of the fact that PCA2501 is a foreign substance whose antisense oligonucleotide is expressed in normal cells.
  • Other viral vectors include adenovirus vectors, HIV (human immunodeficiency virus) vectors, and adeno-associated virus vectors (AAV, adeno-associated virus), and simple virus-less virus (AAV, adeno-associated virus).
  • HS V HIV
  • Epstein-Barr virus (EBV) vector Epstein-Barr virus
  • Non-viral gene transfer methods include calcium phosphate coprecipitation; fusion of ribosomes encapsulating DNA with inactivated Sendai virus whose genes have been disrupted in advance by ultraviolet light to produce membrane-fused ribosomes, which are directly fused with cell membranes.
  • the above-described ligand-DNA complex method includes, for example, a method using asia ligand glycoprotein as a ligand targeting asia mouth glycoprotein receptor expressed by hepatocytes [Wu, et al., J. Biol. Cem., 266 , 14338 (1991); Ferkol, et al., FASEB J., 7, 1081-1091 (1993)) and a method using transferrin as a ligand targeting transferrin receptor strongly expressed in tumor cells [Wagner et al. al., Proc. Natl. Acad. Sci., USA., 87, 3410 (1990)].
  • the gene transfer method used in the present invention may be a combination of various biological and physical gene transfer methods as described above.
  • Examples of the method by the combination include a method of combining a plasmid DNA of a certain size with a polylysine-conjugated antibody specific to an adenovirus / hexon protein.
  • the antisense oligonucleotide of the present invention can be introduced by binding the obtained complex to an adenovirus vector and infecting the cell with the thus obtained trimolecular complex. This method allows for efficient binding, internalization, and endosome degradation before the DNA coupled to the adenovirus vector is damaged.
  • the ribosome ZDNA complex can mediate gene transfer directly in vivo.
  • the retroviral vector system consists of a viral vector and helper cells (packaging cells).
  • the helper cells are the retroviral structural proteins g ag (structural protein in the virion), p o1 (reverse transcriptase), and e n
  • V envelope protein
  • virus vectors have packaging signals and LTRs (long terminal repeats) but do not have structural genes such as gag, po1, and env required for virus replication.
  • the packaging signal is a sequence that serves as a tag during the assembly of the virus particles.
  • the selected gene no, hyg
  • the desired introduced antisense oligonucleotide PCA2501 corresponding to PCA2501
  • PCA2501 corresponding to PCA2501
  • a sense oligonucleotide or a fragment thereof is inserted in place of the viral gene.
  • the viral structural proteins produced by the helper cells package the vector-genomic RNA and form virus particles. Secreted. After the virus particle as a recombinant virus infects target cells, the DNA reversely transcribed from the viral genome RNA is integrated into the cell nucleus, and the antisense gene inserted into the vector is expressed.
  • a method for increasing the efficiency of introduction of a desired gene a method using a fragment containing a cell attachment domain of fibronectin, a heparin binding site and a junction segment CHanenberg, H., et al., Exp.Hemat., 23 , 747 (1995) 3.
  • One of the vectors used in the retroviral vector system is, for example, a retrovirus CMcLachlin, JR, et al., Proc. Natl. Acad. Res. Molec. Biol., Derived from mouse leukemia virus. 38, 91-135 (1990)].
  • the method of using an adenovirus vector will be described in detail.
  • the production of the adenovirus vector is performed by the method of Noknell [Berkner, K.L., Curr.
  • the E1 and / or E3 gene region of the adenovirus early gene is first removed.
  • the desired exogenous gene expression unit (the desired introduced antisense oligonucleotide, ie, the PCA2501 of the present invention, the antisense oligonucleotide, a promoter for transcribing the antisense oligonucleotide).
  • a plasmid vector containing a portion of the adenovirus genome and a plasmid containing the adenovirus genome for example, simultaneously transfected into 293 cells. ⁇ I will do it.
  • the vector of the present invention includes a desired PCA2501 antisense oligonucleotide by causing homologous recombination between the two and replacing the gene expression unit with E1.
  • a non-competent adenovirus vector can be generated.
  • a 3 'adenovirus vector having a terminal protein added thereto can be prepared by incorporating adenovirus genomic DNA into a cosmid vector.
  • a YAC vector can also be used to construct a recombinant adenovirus vector.
  • AAV adeno-associated virus
  • ITR inverted terminal repeat
  • Recombinant AAV can be prepared by utilizing the property of AAV to be integrated into chromosome DNA, and thus a desired gene transfer vector can be prepared. More specifically, this method first leaves the ITRs at both the 5 'and 3' ends of the wild-type AAV, and inserts the desired antisense oligonucleotide (PCA 2501 antisense oligonucleotide) between them. Create a plasmid (AAV vector plasmid). On the other hand, virus proteins required for virus replication and virus particle formation are supplied by another helper plasmid. It is necessary to ensure that there is no common base sequence between the two and that no wild-type virus is produced by genetic recombination.
  • PCA 2501 antisense oligonucleotide desired antisense oligonucleotide
  • both plasmids are introduced by transfection into, for example, 293 cells, and further infected with adenovirus (or non-proliferative type when 293 cells are used) as a helper virus.
  • Recombinant AAV is produced.
  • the cells are recovered by freezing and thawing, and the contaminating adenovirus is inactivated by heating at 56 ° C.
  • the recombinant AAV is separated and concentrated by ultracentrifugation using cesium chloride. As described above, a desired recombinant AV for gene introduction can be obtained.
  • the EBV vector can be produced, for example, according to the method of Shimizu et al. [Norio Shimizu, Cell Engineering, 14 (3), 280-287 (1995)].
  • Epstein-Barr virus was derived from Burkitt's lymphoma in 1964 by Epstein et al. [Kieff, E. and Liebowitz, D .;
  • the EBV genome near the target DNA into which the desired foreign gene is to be integrated is cloned.
  • the DNA fragment of the exogenous gene and the drug resistance gene are then inserted into the vector to produce a recombinant virus.
  • the vector for recombinant virus production cut out with an appropriate restriction enzyme is transfected into EBV-positive Akata cells.
  • Recombinant virus generated by homologous recombination can be recovered together with wild-type Akat EBV by stimulating virus production by anti-surface immunoglobulin treatment.
  • EBV-negative Ak ata cells and selecting a resistant strain in the presence of the drug Ak ata cells infected with only the desired recombinant virus free of wild-type EBV can be obtained.
  • a large amount of the desired recombinant viral vector can be produced.
  • Production of a non-viral vector that introduces a desired antisense oligonucleotide into a target cell without using a recombinant viral vector can be performed, for example, by a gene transfer method using a membrane fusion liposome.
  • membrane ribosomes vesicles composed of lipid bilayer membranes
  • Introduction of the antisense oligonucleotide by the membrane fusion ribosome can be performed, for example, by the method of Nakanishi et al. [Nakanishi, M., et al., Exp. Cell.
  • the contents of the ribosome are introduced into the cells, and the desired antisense oligonucleotide can be introduced into the target cells.
  • the lipid used as the ribosome it is preferable to use a monolayer ribosome having a diameter of 30 O nm made of 50% (molar ratio) cholesterol, lecithin and a synthetic phospholipid having a negative charge.
  • a method for introducing an antisense oligonucleotide into a target cell using another ribosome a method for introducing an antisense oligonucleotide using a cationic ribosome can be mentioned.
  • This method can be carried out according to the method of Yagi et al. [Yagi, K., et al., BBRC, 196, 1042-1048 (1993)].
  • This method focuses on the fact that both the plasmid and the cell are negatively charged, giving a positive charge to both the inner and outer surfaces of the ribosome membrane, increasing plasmid uptake by static electricity and enhancing interaction with the cell. It is assumed that.
  • the ribosome used here is a multilamellar large vesicles (MLV) with a positive charge, but large unilamellar vesicles (LUV) or small unilamellar ribosomes (LUV) are useful. It is also possible to introduce a desired antisense oligonucleotide by forming a complex with a plasmid using small unilamellar vesicles (SUV).
  • MMV multilamellar large vesicles
  • LUV large unilamellar vesicles
  • LUV small unilamellar ribosomes
  • the method of preparing plasmid-embedded catholic MLV is summarized as follows.Firstly, it is TMA (Na-trimethylammonioacetyl) -didodecyl-D-glutamate chloride), DLPC (dilauroyl phosphatidylcholine) and DOPE.
  • TMA Na-trimethylammonioacetyl
  • DLPC diilauroyl phosphatidylcholine
  • DOPE dioleoyl pospatidylethanolamine
  • an expression plasmid incorporating an antisense oligonucleotide for expression in an amount of 0.6 g as a DNA amount in the above-described catonic MLV A method of embedding liposomal lipid to a concentration of 3 Onmol, suspending it in 21 phosphate buffered saline, and administering it to target cells or patient tissues extracted from patients every other day can be exemplified.
  • gene therapy is defined in the Ministry of Health and Welfare guidelines as "administering a gene or a cell into which a gene is introduced into a human body for the purpose of treating a disease".
  • the gene therapy in the present invention means, in addition to the definition of the guideline, the introduction of an antisense oligonucleotide characterized as a PCA2501 expression-suppressing antisense DNA of the PCA2501 antisense oligonucleotide into the aforementioned target cells.
  • an antisense oligonucleotide characterized as a PCA2501 expression-suppressing antisense DNA of the PCA2501 antisense oligonucleotide into the aforementioned target cells.
  • the method of introducing a desired gene into a target cell or target tissue typically includes two types of methods.
  • the cells are cultured outside the body in the presence of, for example, interleukin-2 (IL-12), and retroviruses are added.
  • IL-12 interleukin-2
  • This is a method (ex vivo method) of introducing the desired PCA2501 contained in a virus vector into an antisense oligonucleotide and then re-transplanting the obtained cells. It has been reported that the method is suitable for treating ADA deficiency, genetic diseases caused by defective genes, arteriosclerosis, cancer, AIDS, and the like.
  • the second method is a direct gene transfer method in which the target antisense oligonucleotide (PCA2501 antisense oligonucleotide) is directly injected into the patient's brain or a target site such as lung, liver, or small intestine. Law).
  • PCA2501 antisense oligonucleotide PCA2501 antisense oligonucleotide
  • the first method of the gene therapy is performed, for example, as follows. That is, mononuclear cells collected from a patient are collected from monocytes using a blood separator, and the collected cells are cultured for about 72 hours in an appropriate medium such as AIM-V medium in the presence of IL-12. A vector containing the antisense oligonucleotide to be introduced (PCA2501 antisense oligonucleotide) is added. To increase the transfection efficiency of antisense oligonucleotides, centrifuge at 32 ° C for 1 hour and 2500 rpm in the presence of prominin, then at 37 ° C under 10% CO2 For 24 hours.
  • PCA2501 antisense oligonucleotide PCA2501 antisense oligonucleotide
  • the cells are further cultured for 48 hours in an AIM-V medium or the like in the presence of IL-12, the cells are washed with physiological saline, the number of living cells is counted, and the antisense oligonucleotide
  • the transfection efficiency can be measured by the in situ PCR or, for example, by measuring the degree of the desired antisense and oligonucleotide transfection by measuring the degree of the PCA 2501 activity if the desired target is -as in the present invention. Confirm.
  • the degree of the activity is based on the PCA2501 activity, that is, based on overexpression of the PCA2501 gene, such as brain dopamine 1, dopamine 2, noradrenaline, serotonin (5-HT), acetylcholine, etc.
  • the degree of the overproduction activity of the sympathetic stimulant, the activity of stimulating the sympathetic neurotransmitter receptor, and the activity of these neurotransmitters that cause the impaired transmission may be measured.
  • the safety level is checked by culturing bacteria and fungi in cultured cells, detecting the presence of mycoplasma infection, searching for endotoxin, etc.
  • the cultured cells into which the effective dose of antisense oligonucleotide (PCA2501 antisense oligonucleotide) has been introduced are returned to the patient by intravenous drip.
  • Gene therapy is performed by repeating such a method at intervals of, for example, weeks to several months.
  • the dose of the viral vector is appropriately selected depending on the target cells to be introduced.
  • a virus-producing cell containing a retroviral vector containing the desired antisense oligonucleotide (PCA2501 antisense oligonucleotide) is co-cultured with, for example, a patient's cell. It is also possible to adopt a method of introducing an antisense oligonucleotide (PCA2501 antisense oligonucleotide) into a cell.
  • the target antisense oligonucleotide (PCA2501 antisense oligonucleotide) is actually introduced by the gene transfer method, especially through preliminary experiments in vitro.
  • the desired treatment is based on the search for the vector gene cDNA by PCR or in situ PCR in advance, or the desired treatment based on the introduction of the desired antisense oligonucleotide (PCA2501 antisense oligonucleotide). It is desirable to confirm the effects such as an increase in specific activity, an increase in growth of target cells, and suppression of growth.
  • peripheral blood cells PCA2501-expressing cells
  • the enzyme After treatment, the cultured cells are established, and then introduced into peripheral blood cells (PCA2501-expressing cells) targeting the desired antisense oligonucleotide using, for example, a retrovirus.
  • peripheral blood cells PCA2501-expressing cells
  • the expression levels of IL-112 etc. are measured (in vivo), and then radiation treatment is performed to inoculate the patient's peripheral blood, lung, liver, small intestine or brain.
  • radiation treatment is performed to inoculate the patient's peripheral blood, lung, liver, small intestine or brain.
  • the present invention also provides, as an active ingredient, a cell into which the antisense oligonucleotide introduction vector or the intended antisense oligonucleotide (PCA2501 antisense oligonucleotide, etc.) of the present invention has been introduced.
  • a pharmaceutical composition (gene therapeutic agent) containing a pharmaceutically effective amount of the compound and a suitable nontoxic pharmaceutical carrier or diluent.
  • Pharmaceutical carriers that can be used in the pharmaceutical composition (pharmaceutical formulation) of the present invention include fillers, bulking agents, binders, humectants, disintegrants, surfactants, which are usually used according to the use form of the formulation. Diluents and excipients such as lubricants can be exemplified, and these can be appropriately selected and used depending on the dosage unit form of the obtained product.
  • the dosage unit form of the pharmaceutical preparation of the present invention As the dosage unit form of the pharmaceutical preparation of the present invention, the above-mentioned preparation examples of the PCA2501 protein antibody preparation can be similarly mentioned, and it can be appropriately selected from various forms according to the purpose of treatment.
  • a pharmaceutical preparation containing the vector for introducing an antisense oligonucleotide of the present invention can be used in a form in which the vector is embedded in ribosomes or a virus containing a retrovirus vector containing a desired antisense oligonucleotide.
  • the vector is embedded in ribosomes or a virus containing a retrovirus vector containing a desired antisense oligonucleotide.
  • it is prepared in the form of infected cultured cells.
  • PH 7.4 phosphate buffered saline
  • Ringer's solution injection for intracellular composition
  • a substance that enhances gene transfer efficiency such as protamine.
  • It can also be prepared in such a form that it is administered together with it.
  • the amount of the active ingredient to be contained in the above pharmaceutical preparation and the dose thereof are not particularly limited, and may vary widely depending on the desired therapeutic effect, administration method, treatment period, patient age, gender and other conditions. It is appropriately selected.
  • the dosage of the desired antisense O Rigo nucleotide-containing Leto Rowirusu base Kuta one as a pharmaceutical formulation per day of body weight l kg per, for example, about 1 X 10 3 pfu mosquito et 1 X as mosquito-valent Retro Virus It should be about 10 15 pfu.
  • the preparation may be administered once or several times a day, or may be administered intermittently at intervals of one to several weeks. Preferably, it can be co-administered with a substance that enhances gene transfer efficiency, such as promin, or a preparation containing the same.
  • the gene therapy according to the present invention When the gene therapy according to the present invention is applied to the treatment of schizophrenia, the above-mentioned various gene therapies can be appropriately combined (combined gene therapy). Pharmacotherapy, occupational therapy, etc. can be combined. Furthermore, the gene therapy of the present invention, including its safety, can be performed with reference to the guidelines of NIH [Recombinant DNA Advisory Committee, Human Gene Therapy, 4, 365-389 (1993)].
  • a biological sample such as a tissue or a body fluid (for example, blood or serum) is prepared, and if necessary, nucleic acid is extracted to detect the presence of the PCA2501 gene. It is possible to analyze whether or not.
  • the detection method includes, for example, preparing a PCA2501 DNA fragment,
  • PCR polymerase chain reaction
  • a primer having the same sequence as PCA2501 is prepared, used as a screening probe, and reacted with a biological sample (nucleic acid sample) to confirm the presence of the gene having the PCA2501 sequence.
  • the nucleic acid sample may be prepared by various methods that facilitate detection of the target sequence, for example, denaturation, restriction digestion, electrophoresis or dot blotting.
  • the PCR method is particularly preferred in terms of sensitivity.
  • the method is not particularly limited as long as the method uses the PCA2501 fragment as a primer. , 1350-1354 (1985)) and a newly developed or future modified PCR method (Yoshiyuki Sakaki, et al., Ed., Yodosha, Experimental Medicine, extra edition, 8 (9) (1990); Proteins and nucleic acids ⁇ Enzymes, extra editions, Kyoritsu Shuppan Co., Ltd., '35 (17) (1990)) can be used.
  • the DNA fragment used as a primer is a chemically synthesized oligo DNA, and these oligo DNAs are synthesized using an automatic DNA synthesizer or the like, for example, a DNA synthesizer (Pharmacia LKB Gene Assembler Plus: manufactured by Pharmacia). can do.
  • the length of the synthesized primer is preferably about 10 to 30 nucleotides.
  • a probe used for the above-mentioned screening is usually a labeled probe, but may be unlabeled or may be detected by specific binding to a directly or indirectly labeled ligand.
  • Suitable labels as well as methods for labeling probes and ligands, are known in the art and can be incorporated by known methods, such as nick translation, random priming, or kinase treatment. These techniques include radioactive labels, biotin, fluorescent groups, chemiluminescent groups, enzymes, antibodies, and the like.
  • PCR method used for the detection for example, an RT-PCR method is exemplified, and various modifications used in the art can be applied.
  • the measurement method of the present invention can be easily carried out by using a reagent kit for detecting the PCA2501 gene in a sample.
  • the present invention provides a reagent kit for detecting PCA2501, comprising the PCA2501 DNA fragment.
  • the reagent kit contains, as an essential component, a DNA fragment that hybridizes to at least a part or all of the nucleotide sequence shown in SEQ ID NO: 2 or its complementary nucleotide sequence, a labeling agent may be used as another component.
  • reagents essential for the PCR method for example, TaqDNA polymerase, deoxynucleotide diphosphate, primer, etc. may be contained.
  • the labeling agent examples include a chemical modifying substance such as a radioisotope or a fluorescent substance, and the DNA fragment itself may be conjugated with the labeling agent in advance.
  • the reagent kit may contain a reaction diluent, a standard antibody, a buffer, a detergent, a reaction stop solution, etc., which are suitable for the benefit of performing the measurement.
  • the present invention also provides a method for diagnosing schizophrenia using the above-mentioned measurement method, a diagnostic agent and a diagnostic kit used for the method.
  • the present invention also provides a method for screening related genes related to the human PCA2501 gene in a test sample by such measurement and sequencing of the PCA2501 DNA in the test sample.
  • a protein encoded by the human PCA2501 gene represented by SEQ ID NO: 1 of the present invention or one or several to a plurality of amino acids in SEQ ID NO: 1
  • synthesizing a protein from the deleted, substituted or added amino acid sequence, or a fragment thereof, or synthesizing an antibody against the protein wild type PCA2501 and / or mutant PCA2501 can be measured.
  • the present invention provides a method for assaying an antibody and an antigen for wild-type PCA2501 and / or mutant PCA2501.
  • this method it is also possible to detect the degree of schizophrenia disorder or the degree of schizophrenia progression based on a change in wild-type PCA2501 polypeptide.
  • Such alterations can also be determined by PCA 2501 sequence analysis by conventional techniques in the art, but more preferably by using antibodies (polyclonal or monoclonal antibodies) to determine differences in the PCA 2501 protein or PCA 2501 protein. Can be detected.
  • the PCA2501 antibody is obtained by immunoprecipitating the PCA2501 protein from a solution containing a biological material sample collected from a human such as blood or serum, and Western plotting a polyacrylamide gel. Alternatively, it can react with the PCA2501 protein on an immunoblot.
  • the PCA2501 antibody can also detect PCA2501 protein in paraffin or frozen tissue sections using immunohistochemical techniques.
  • More preferred examples related to methods for detecting wild-type PCA 2501 or a mutant thereof include enzyme-linked immunosorbent assays (ELI), including sandwich methods using monoclonal antibodies and Z or polyclonal antibodies. SA), radioimmunoassay (RIA), immunoradiometric assay (IRMA), and immunoenzymatic assay (IEMA).
  • ELI enzyme-linked immunosorbent assays
  • SA enzyme-linked immunosorbent assays
  • RIA radioimmunoassay
  • IRMA immunoradiometric assay
  • IEMA immunoenzymatic assay
  • the present invention can also provide a PCA2501 receptor present on the cell membrane fraction or the cell surface, which has a PCA2501 binding activity to the PCA2501 protein.
  • Acquisition of the PCA2501 receptor includes the cell membrane fraction This is achieved by conjugating the labeled PCA2501 protein in a biomaterial sample, extracting, isolating and purifying a PCA2501 binding reaction, and specifying the amino acid sequence of the isolated product, thereby obtaining the PCA2501 receptor protein.
  • sequencing can be readily accomplished by those skilled in the art.
  • the present invention provides a method for screening a PCA2501 receptor protein or a binding fragment thereof for any of various drugs by using the compound (PCA2501 receptor protein: a low molecular compound, a high molecular compound, Protein, partial protein fragment, antigen, or antibody).
  • PCA2501 Reception is used.
  • the PCA2501 receptor polypeptide or a fragment thereof used in such a screening test may be a free substance in a solution attached to a solid support or transported to the cell surface.
  • Examples of drug screening include, for example, the use of prokaryotic or eukaryotic host cells stably transformed with a recombinant protein expressing the PCA2501 protein or a fragment thereof, preferably in a competitive binding assay. it can. Such cells in free or fixed form can also be used for standard binding assays. More specifically, the formation of a complex between the PCA2501 receptor protein or fragment thereof and the substance to be tested is measured, and the complex between the PCA2501 receptor protein or fragment thereof and the PCA2501 protein or fragment thereof is measured. Compounds can be screened by detecting the extent to which body formation is inhibited by the substance being tested.
  • the present invention provides for the use of such materials with PC A2 by methods known in the art.
  • the present invention can provide a drug screening method characterized by measuring the presence of a complex between a 2501 receptor protein or a fragment thereof and a ligand. You. In addition, the activity of the PCA2501 receptor was measured and the substance was identified as PCA2
  • PCA2501 a sympathetic nerve stimulating action such as dopamine, a receptor stimulating action such as a dopamine receptor, or a sympathetic stimulating action such as noradrenaline? Or whether it is possible to regulate protein-protein interaction or to control complex formation.
  • a sympathetic nerve stimulating action such as dopamine
  • a receptor stimulating action such as a dopamine receptor
  • a sympathetic stimulating action such as noradrenaline
  • the 2501 receptor protein or a fragment thereof is separated from that present in the protein: protein complex, and the amount of free (uncomplexed) label is determined by the P
  • a small peptide (peptide mimetic) of the PCA2501 protein can be analyzed in this way, and the PCA2501 receptor inhibitory activity can be measured (b).
  • another method for drug screening is a screening method for compounds having an appropriate binding affinity for the PCA2501 receptor protein, which, in short, may comprise a number of different peptide test compounds. Is synthesized on a solid support, such as the surface of a plastic pin or other material, and then the peptide test compound is reacted with the PCA2501 receptor protein and washed. Next, a method of detecting the reaction-bound PCA 2501 receptor protein using a known method can be exemplified (PCT Patent Publication No. WO 84-03564). Purified PCA2
  • the 501 receptor can be coated directly on plates used in the drug screening techniques described above.
  • the PCA2501 receptor protein can be immobilized on a solid phase by supplementing the antibody with a non-neutralizing antibody against the polypeptide.
  • the present invention is also directed to the use of a competitive drug screening assay to determine the binding to the PCA2501 receptor protein or a fragment thereof.
  • a neutralizing antibody capable of specifically binding to the PCA2501 receptor protein and a test compound To compete.
  • the competition by the antibodies makes it possible to detect the presence of any peptide having one or more antigenic determinants of the PCA2501 receptor protein. .
  • a PCA2501 gene expression product or a PCA2501 protein (hereinafter, also referred to as PCA2501 protein), or an antibody against fragments thereof
  • a test solution and a labeled PCA2501 protein, a partial peptide of the PCA2501 protein or a salt thereof in a competitive manner, and a labeled PCA2501 protein or a partial peptide of the PCA2501 protein bound to the antibody is reacted.
  • a screening method for quantifying a PCA2501 protein, a partial peptide of PCA2501 protein or a salt thereof in a test solution, which comprises measuring the ratio of a tide or a salt thereof, is also possible (b).
  • the PCA2501 protein, the PCA2501 protein when the substrate is brought into contact with the partial peptide of the PCA2501 protein or a salt thereof and the PCA2501 protein, the PCA2501 protein when the substrate and the test compound are brought into contact with the partial peptide of the PCA2501 protein or a salt thereof By measuring and comparing the activity of partial peptides of PCA2501 protein or their salts, compounds or salts thereof that inhibit the activity of PCA2501 protein or its salts (eg, PCA2501 activity) can be screened. It is possible (see d below).
  • a screening method for identifying a compound that stimulates or suppresses the function of a PCA2501 polypeptide or a PCA2501 gene expression product is provided.
  • A binding of the candidate compound to the polypeptide or gene expression product (or the cell carrying the polypeptide or the gene expression product or its membrane) or a fusion protein thereof;
  • B a method of measuring by a label directly or indirectly bound to a compound;
  • the candidate compound is the polypeptide or Whether the activation or repression of the gene expression product results in a signal or not is determined by the cell or cell membrane carrying the polypeptide or gene expression product.
  • additional methods include the use of host eukaryotic cell lines or cells that contain a non-functional PCA2501 gene. After a host cell line or cell is grown for a period of time in the presence of the drug compound, the rate of growth of the host cell is measured to determine whether the compound is capable of regulating cell growth, or Confirm whether the ability to regulate mutual binding or complex formation can be controlled. As one means of measuring the growth rate, the biological activity of the PCA 2501 receptor can be measured.
  • a more active or stable form of the PCA2501 protein derivative or, for example, an agent that enhances or interferes with the function of the PCA2501 protein in vivo The organisms with which they interact to develop It is possible to produce a chemically active protein or structural analog, for example, a PCA2501 agonist, a PCA2501 antagonist, a PCA2501 inhibitor, and the like.
  • the structural analog can be determined by, for example, X-ray crystallography, computer modeling, or a combination of these methods for determining the three-dimensional structure of a complex of PCA 2501 and another protein.
  • information on the structure of a structural analog can be obtained by modeling a protein based on the structure of a homologous protein.
  • analysis can be performed by alanine 'scan.
  • the method involves substituting Ala for an amino acid residue and analyzing each amino acid residue of the peptide in this way by measuring its effect on the activity of the peptide, and identifying regions important to the activity and stability of the peptide. How to decide.
  • a more active or stable PCA2501 derivative can be designed.
  • PCA2501 activity or stability or action as an inhibitor, agonist, antagonist or the like of PCA2501 activity can be designed and developed.
  • This method is a technique for intentionally modifying the genetic information of an organism by utilizing homologous recombination of genes, and can be exemplified by a method using mouse embryonic stem cells (ES cells) (Capeccchi, M. et al. , Science, 244, 1288-1292 (1989)).
  • ES cells mouse embryonic stem cells
  • a mutant mouse can be easily prepared by adapting the human wild-type PCA2501 gene and the mutant PCA2501 gene of the present invention. Therefore, by applying the above technology, it is possible to design and develop a drug having improved PCA2501 activity or stability or an action as an inhibitor, agonist, antagonist or the like of PCA2501 activity.
  • the mRNA extracted from peripheral blood mononuclear cells of the treatment group (7 cases), the non-treatment group (3 cases), and the healthy subjects (11 cases) was determined by the conventional method.
  • the synthesized cDNA was amplified by P CR in the presence of anchored primer - [ ⁇ - 33 P] ATP 3 were labeled with (Amersham) '. PCR amplification of this cDNA was performed as follows.
  • 25 1 PCR mixed solution for 21 cDNAs was obtained by mixing 2 1 single reaction mixture, 2 1 10 XPCR buffer (Yukara), and 2.5 mM dNTP. s, 0. 25 1 of £ Ding & (1 DN a polymerase Ichize (5 units Zm 1: evening made Kara Co., Ltd.), 1 1 of [ ⁇ - 33 P] 3 of 25pmol labeled with ATP '- anchor over de ⁇ Oligo-25 pmol of dT primer and 11 1, 5-primer-1 primer (29 primers, SEQ ID NO: 4: 5 '-CTGATCCATG-10-mer with an arbitrary sequence of the sequence shown in 3' (Deoxyoligonucleotide primer) and 10.251 distilled water
  • the PCR reaction was performed under the following conditions: 95 ° C for 5 minutes followed by 95 ° C For 3 minutes, 40 ° C for 5 minutes and 72 ° C for 5 minutes, then 95 for 0.5 minutes, 25 cycles of 40 ° C for 2 minutes and 72 ° C for 1
  • the PCR reaction sample was extracted with ethanol, resuspended in formamide sequencing dye, applied on a 6% acrylamide, 7.5 M rare-sequencing gel, and subjected to electrophoresis.
  • the gel was dried without fixing, and autoradiography was performed.
  • a radioactive ink is marked on a 3 MM filter paper on which a dried gel has been placed in advance, and by combining this with the autoradiogram, the target schizophrenic patient group (dosing group, unmedicated)
  • the specific band containing the cDNA expressed in both groups was cut out from the dried gel together with 3 MM filter paper, and stirred for 1 hour at 3001 dH 2 ⁇ .
  • cDNA and in the presence of 0. 3M NaOAc and 1 1 of 1 OmgZm 1 glycogen as a carrier again collected by ethanol precipitation and redissolved in dH 2 0 10 1.
  • 51 of this solution was used to re-amplify the target fragment by PCR under the same conditions as the first time.
  • the PCR product was purified from the band similarly excised from the gel, and a third PCR was performed under the same conditions.
  • the PCR product is subcloned into the Hinc II site of pUC118 vector (Yukara) and the base sequence is sequenced by ABI377 auto-sequencer (Applied Biosystems). Were determined.
  • This product consists of 98 nucleotides and was named PCA2501.
  • the human normal heart cDNA library (Stratagene) is composed of oligo (dT) + random hexoma-11-primed human normal heart cDNA and Uni-ZAP tmX.
  • the present inventors confirmed about 5 plaques for PCA2501. From these results, the amount of transcription between the total RNAs was calculated to be about 0.00001%.
  • primers were designed based on the nucleotide sequence of the gene fragment, and the 5'-RACE (Rapid Amplification of cDNA End) method (MA From man, et al., Proc. Natl. Acad. Sci., USA., 8, 8998 (1988)). That is, the isolation and analysis of the cDNA clone containing the 5 'part of the gene of the present invention was performed by partially modifying the protocol for using the product, and using a commercially available kit (5'-Rapid AmpliFinder RACE Kit, Clontech). According to the method, it was isolated as follows.
  • the gene-specific primers P1 and primer P2 used here were synthesized according to a conventional method, and their base sequences are shown in Table 2 below (P1 is shown in SEQ ID NO: 5 and P2 is shown in SEQ ID NO: 6) As shown in FIG.
  • the anchor and primer used were those attached to a commercially available kit.
  • P2 primer 5,-CCATCAGTTTTTCCAATGTGA-3
  • a human normal heart and lung cDNA library (Marathon-Ready cDNA: manufactured by Clontech) as type II, the cDNA was amplified by PCR using P1 primer and P2 primer.
  • the PCR reaction conditions were: 95 ° C for 2 minutes, followed by a cycle of 95 ° C for 30 seconds and 68 ° C for 4 minutes.
  • the PCR product was analyzed by 1.5% agarose gel electrophoresis.
  • a band with a size of about 1000 bases was detected by agarose gel electrophoresis, and the product of this band was inserted into pT7B1ueT vector (Novagen), and insertion of an appropriate size was observed. Several clones were selected.
  • PCA2501 5-1 and PCA2501—15 Sequences encoding the protein, 5 ′ and 3 ′ flanking sequences and full length by sequencing cDNA clones The sequence of 3910 bases was determined, the gene was named schizophrenia-related gene (PCA2501 gene), and the DNA sequence thus obtained was named PCA2501.
  • This PCA2501 cDNA contains an open reading frame of the 2157 nucleotide sequence represented by SEQ ID NO: 2 which encodes a protein consisting of the 718 amino acid sequence represented by SEQ ID NO: 1 having a calculated molecular weight of 77972 Da. It contained the full-length 3910 nucleotide sequence shown in 3. In the sequence shown in SEQ ID NO: 3, the position of the start codon was ATG at position 99 to position 101 of the sequence number, and the position of the stop codon was TAG at position 2253 to position 2255.
  • Example 1 For Northern blot analysis, human MTN (Multiple-Tissue Northern) blots I and II (Clontech) were used. cDNA fragments, T3 and T7 using primers' set of promoter sequences, PCR by [ ⁇ one 33 P] prehybridized da I's The membrane containing the PCR amplification product obtained in Example 1 were labeled in one d CTP (The conditions were in accordance with the product protocol), and then hybridization was performed in accordance with the product protocol.
  • human MTN Multiple-Tissue Northern
  • T3 and T7 using primers' set of promoter sequences
  • PCR by [ ⁇ one 33 P] prehybridized da I's
  • the membrane containing the PCR amplification product obtained in Example 1 were labeled in one d CTP (The conditions were in accordance with the product protocol), and then hybridization was performed in accordance with the product protocol.
  • the human tissues used were heart (heart), brain (brain), placenta (Placenta), lung (Lung), liver (Liver), skeletal muscle (S. muscle), kidney (Kidney), kidney (Pancreas), They are spleen (Spleen), thymus (Thymus), prostate (Prostate), testis (Testis), ovary (Ovary), small intestine (Small intestine), colon (Colon) and peripheral blood leukocytes (PBL).
  • a 4 kb transcript homologous to PCA2501 was expressed in the heart, placenta, lung, liver, ⁇ ⁇ spleen, small intestine, peripheral blood, and lymphocytes, but the level of expression was not so significant.
  • Radiation hybrid mapping allows you to The localization of the chromosome of the loan was determined (Cox, DR, et al., Science, 250, 245-250. That is, two types of primers were designed from the nucleotide sequence of the gene fragment obtained by the DD method, I purchased a 4-radiation hybridization panel (GeneBridge4; manufactured by Research Genetics), and according to the manufacturer's instructions, used this as a template to set the bases shown in SEQ ID NO: 5 and SEQ ID NO: 6. PCR (30 cycles) was performed using the P1 and P2 primers of the sequence, and a PCR reaction for chromosome mapping analysis was performed.
  • the PCA2501 gene was located at the marker AFM126ZC5. Since this marker is a marker localized at 3q11-q12 on the chromosome, it was confirmed that PCA2501 was localized at chromosome position 3q11-q12. .
  • DNA competitors for PCA2501 and GAPDH were prepared using a competitive DNA construction kit.
  • TaKaRa (Yukara Co., Ltd .: TaKaRa) was used to perform PCR with ADNA as type II DNA, and DNA competition of PCA2501 and GAPDH (housekeeping gene) was performed using SUPREC-2 (Takara). Refine Yuichi each did.
  • the fission products were separated on a 1.5% agarose gel and stained with ethidium bromide.
  • the desired PCA2501 Competition Yuichi (116bp) and GAPDH Competition Yuichi (400bp) were recovered from the gel using SUPREC-2 (Yukara).
  • the absorbance (0D 26 fl ) of the purified DNA competitor was measured, and the number of copies was calculated by the following formula.
  • cDNA was synthesized from 5 g of the purified total RNA using Superscript II TM RNase H-reverse transcriptase (Life's Technology) using oligo d (T). Each resulting cDNA product was used for competitive PCR amplification.
  • the PCR reaction consisted of 25 1 final volumes of 2.5 ng of 50 ng Z1 cDNA from each of the above samples, 2.51 DNA competitors (10 3 , 10 4 , 10 5 , 10 6 , 1 0 7, 1 0 8 copies / 2. 5 / xl), 2. 1 OX buffer 5 1 (manufactured by T0Y0B0 companies), 2. 5 l of 2.
  • PCR reaction conditions were 95 ° C, 2 minutes, followed by 95 ° C, 0.5 minutes, 60 ° (30 minutes, 0.5 minutes, 75 ° C, 0.5 minutes).
  • the amplified product was 98 base pairs for the PAC2501 gene.
  • GAPDH sense 'primer SEQ ID NO: 13
  • GAPDH antisense primer SEQ ID NO: 1
  • the reaction was performed at 95 ° C, 0.5 min, 55 ° (:, 0.5 min, 75 ° C, 0.5 min under 30 cycles.
  • the amplified product was reacted against GAPDH.
  • Each PCR product thus obtained was subjected to 5% polyacrylamide gel electrophoresis. Separated and dried on a filter paper using a gel dryer. After drying, exposure to an image plate was performed, and image data was captured using a Molecular Imager GS-525 (Molecular Imager GS-525: manufactured by Bio-Rad). Data analysis was performed using a molecular analyst (Molecular Analyst: manufactured by Pio Rado).
  • the candidate gene PCA 2501 was significantly expressed in schizophrenic patients compared to healthy subjects or non-schizophrenic patients.
  • the isolated PCA2501 gene of the present invention is specifically highly expressed in schizophrenic patients, and is useful for diagnosis of schizophrenic patients.
  • the psychiatric symptoms were evaluated by the PAN SS (Positive and Negative Syndrome Scale; Schizophr. Bull., 13, 261-276 (1987)).
  • the composition scale is the value obtained by subtracting the negative symptom scale from the PAVS S positive symptom scale.
  • PCA2501 gene expression is expressed in peripheral blood mononuclear cells collected.
  • the vertical axis represents the expression level of the PCA2501 gene
  • the horizontal axis represents the composition scale of PANSS.
  • SEQ ID NO: 15 to SEQ ID NO: 28 designed from PCA2501 gene were synthesized (A: SEQ ID NO: 15 and B: SEQ ID NO: 16, C: SEQ ID NO: 17 and D: SEQ ID NO: 18, E: SEQ ID NO: 19 and F: SEQ ID NO: 20, G: SEQ ID NO: 21, and H: SEQ ID NO: 22, I: SEQ ID NO: 23 and J: SEQ ID NO: 24, K: SEQ ID NO: 25 and L: SEQ ID NO: 26, M: SEQ ID NO: 27 and N: SEQ ID NO: 28) These were used for PCR.
  • Fl_, F2-, F3-, N1-, N2-, N3_, and C-PCA 250 1 2173, 2098, 1876, 1300, 1225, 1003, 911 base pairs
  • gene fragments were obtained.
  • the obtained gene fragment was subcloned into PCR 2.1 (Invitrogen).
  • expression vectors pQE31 F, F2-, F3-, N1-, N2-, N3-PCA2501) and pQE30 (C-PCA2501) ( QIAGEN).
  • the resulting vector was transfected into Escherichia coli M15.
  • the transfected E. coli was cultured for 4 hours in the presence of IPTG to recover the E. coli.
  • F1-, F2-, F3_, Nl_, N2-, N3-, and C-PCA 2501 were analyzed by affinity chromatography using Ni-NT A agarose (manufactured by QIAGEN). , 705, 631, 447, 422, 348, 310 amino acid sequences) were purified. Using these as immunogens, anti-PC A2501 antibodies were prepared by a conventional method.
  • the reaction of the anti-PCA2501 antibody with a sample obtained from a patient sample can be used for diagnosis of schizophrenia.
  • PCA2501 gene of the present invention may make it possible to treat schizophrenia using an antibody against PCA2501 and an antisense DNA.
  • Real-time PCR (ABI PRISM (im)
  • PCA2501 7700 Sequence Detection System User's Manual: 5.10.5.13 (1996), Patent Registration No. 2825 976, etc.
  • the expression of PCA2501 can be easily measured by a simple method using this method, and this can be used for diagnosis of schizophrenia.
  • a novel PCA2501 gene which is specifically developed in schizophrenic patients.
  • a detection method and a detection method for detecting schizophrenia by detecting a PCA2501 gene or a gene product in a sample that binds to the oligonucleotide 'probe using the PCA2501 oligonucleotide as a probe Kit is provided.
  • an antibody using the gene expression product as an antigen can be produced.
  • a diagnosis of schizophrenia using the antibody and a pharmaceutical composition and a drug for schizophrenia containing the antibody as an active ingredient.
  • the PCA2501 protein encoded by the gene can be produced in large quantities by genetic engineering. According to the provision of the protein, the PCA2501 protein activity and the binding of Functions such as activity can also be examined.
  • the PCA2501 protein is also useful for elucidating the pathology, diagnosing, and treating diseases involving the PCA2501 gene and its product (eg, schizophrenia and schizophrenia complications).
  • a gene transfer vector useful for gene therapy containing the PCA2501 antisense oligonucleotide, the cell into which the PCA2501 antisense oligonucleotide has been introduced, and the vector or the cell as an active ingredient
  • the present invention provides a gene therapy agent and a gene therapy method using the same.

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Abstract

Cette invention se rapporte à un gène contenant un polynucléotide qui code la séquence d'acide aminé représentée par le numéro d'identification de séquence 1; et au polynucléotide codé par ce gène. Ce gène est associé à la schizophrénie.
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WO2004024069A3 (fr) * 2002-09-11 2005-04-14 Genentech Inc Compositions et methodes pour le traitement de maladies liees au systeme immunitaire

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