WO2003072779A1 - Methode d'utilisation de genes specifiques a l'hypophyse - Google Patents

Methode d'utilisation de genes specifiques a l'hypophyse Download PDF

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WO2003072779A1
WO2003072779A1 PCT/JP2003/002109 JP0302109W WO03072779A1 WO 2003072779 A1 WO2003072779 A1 WO 2003072779A1 JP 0302109 W JP0302109 W JP 0302109W WO 03072779 A1 WO03072779 A1 WO 03072779A1
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pituitary
human
seq
nos
protein
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Ke-Ita Tatsumi
Susumu Tanaka
Nobuyuki Amino
Kousaku Okubo
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Japan Science And Technology Agency
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/10—Drugs for disorders of the endocrine system of the posterior pituitary hormones, e.g. oxytocin, ADH
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to genes and amino acids specifically expressed in the pituitary. Conventional technology
  • the pituitary gland consists mainly of endocrine cells that make pituitary hormones. These hormones are stored in secretory granules and released by regulated exocytosis (extracellular release). These secretory granules also contain processing enzymes that melt into cell membranes and release hormones and other components (Arvan P & Castle D Biochemical Journal 332 593-610 (1998))
  • This characteristic function of the pituitary gland is maintained by proteins such as the pituitary hormone ⁇ transcription factor, which is unique to the pituitary gland. Abnormalities in these genes result in both a deficiency of isolated anterior pituitary hormone and a deficiency of bound pituitary hormone (.Phillips III JA et al. Proceeding of the National Academy of Sciences of the United States of America 78 6372-6375 (1981); Tatsumi K et al. Nature Genetics 1 56-58 (1992); Cushman LJ & Camper, Mammalian Genome 12 485—494 (2001)).
  • Tanno ⁇ 0 click modifying enzymes this ⁇ or GalNAc-4-pituitary as sulfotransferase specifically intended to express (Okuda T et al. Journal of Biological Chemistry 51 40605- 40613 (2000)), prohormone convertase synthetase (PC) 1/3, PC 2, carboxypeptidase E (CPE) and neuroendocrine protein 7B2 (Iguchi H et al. Neuroendocrinology 39 453-458 (1984)) some force s are also expressed in ⁇ (Gorr SU et al. Molecular and Cellular Endocrinology 1721-6 (2001)).
  • GSs gene signatures
  • the expressed genes are identified by their GS species, and the relative abundance of each transcript can be estimated by the frequency of the corresponding GS species in the library.
  • the resulting gene expression profile represents the amount of gene transcription in the human pituitary.
  • a profile of similar gene expression in 64 human tissues constitutes a molecular anatomical database called the Body Map database (Japanese Clinical Journal Vol. 52 No. 4 (4, 1994); Hishiki T et al. Nucleic Acids Research 28 136-138 (2000); Kawamoto S et al. Genome Research 10 1817-1827 (2000)).
  • the Body Map database Japanese Clinical Journal Vol. 52 No. 4 (4, 1994); Hishiki T et al. Nucleic Acids Research 28 136-138 (2000); Kawamoto S et al. Genome Research 10 1817-1827 (2000).
  • the present inventors have identified genes that are abundantly expressed in human pituitary tissues by examining genes that are actively expressed in human pituitary. By comparing the expression profile of this gene in the human pituitary gland with a body map database, we found genes that are specifically expressed in the human pituitary gland and found that they can be used for the detection and treatment of diseases in the human pituitary gland .
  • PGSF 1a (splice variant of GS 9544): SEQ ID NO: 1
  • PGSF lb (splice variant of GS 9544): SEQ ID NO: 2
  • PGSF2 (same as GS 9589): SEQ ID NO: 3
  • the nucleotide sequence and amino acid sequence of pi-a have already been registered (AX127740, AX036667, AK026832.AB037745), PGSFla (AB058892), PGSFlb (AB058893) and PGSF2 (AB058894).
  • the inventors registered S14—APR—2001.
  • the present invention is any one of the nucleotide sequences of SEQ ID NOS: 1 to 3, and it can be said that the present invention is any one of the amino acid sequences of SEQ ID NOS: 5 to 7.
  • the present invention relates to the use of any one of SEQ ID NOs: 1 to 4 or a portion thereof for testing for human pituitary gland or human joint or treating human pituitary gland-related disease or human joint-related disease.
  • the present invention includes the following inventions.
  • a kit for diagnosing a genetic abnormality in a human pituitary-related disease or a human joint-related disease comprising a primer for PCR amplifying any one of the protein coding regions of SEQ ID NOs: 1 to 4. Furthermore, a method for diagnosing a genetic abnormality in a human pituitary-related disease or a human joint-related disease using a primer for PCR amplifying any one of the protein coding regions of SEQ ID NOs: 1 to 4.
  • SEQ ID NO: 1 A diagnostic agent for gene expression of a human pituitary-related disease or a human joint-related disease, using a partial sequence of any one of SEQ ID NOs: 1 to 4 as a probe. Furthermore, SEQ ID NO: 1
  • a method for diagnosing gene expression of a human pituitary-related disease or a human joint-related disease using a partial sequence of any one of the base sequences of any one of (1) to (4) as a probe.
  • a human pituitary-related disease in which low expression of the protein of any of SEQ ID NOs: 5 to 8 is an exacerbation factor using an expression vector containing any of the nucleotide sequences of SEQ ID NOs:! Or a method for treating a human joint-related disease.
  • SEQ ID NOs: 1-4 consisting of an antisense nucleic acid to any of the base sequences
  • SEQ ID NOs: 5-8 a human pituitary-related disease or human whose overexpression of any of the proteins is an exacerbating factor Treatment for joint-related diseases.
  • the present invention relates to a test for a human pituitary gland or a human joint or a treatment for a human pituitary gland-related disease or a human joint-related disease, using a protein having any of SEQ ID NOS: 5 to 8 or an antibody against the protein. Is for use.
  • the present invention includes the following inventions.
  • a diagnostic agent for a human pituitary-associated autoimmune disease comprising a protein having any one of SEQ ID NOs: 5 to 8 as an antigen.
  • a method for diagnosing a human pituitary gland or a human joint-related autoimmune disease using the protein of any one of SEQ ID NOs: 5 to 8 as an antigen.
  • the antigen may be labeled, and the autoimmune disease may be autoimmune pituititis or rheumatoid arthritis.
  • a diagnostic agent for human pituitary function comprising an antibody against a protein having any one of SEQ ID NOs: 5 to 8. Furthermore, a method for diagnosing human pituitary or human joint function using an antibody against a protein having any one of SEQ ID NOs: 5 to 8.
  • a therapeutic agent for a human pituitary-related disease or a human joint-related disease which comprises a protein having any of the sequences of SEQ ID NOS: 5 to 8, wherein low expression of the protein of any of the sequences of SEQ ID NOs: 5 to 8 is an exacerbating factor. Furthermore, treatment of a human pituitary-related disease or a human joint-related disease in which low expression of any of the sequence proteins of SEQ ID NOS: 5 to 8 becomes a worsening factor, using a protein of any of the sequences of SEQ ID NOs: 5 to 8. Method.
  • An agent for treating a human pituitary-related disease which comprises an antibody against a protein having any of the sequences of SEQ ID NOS: 5 to 8, and whose overexpression of a protein having any of the sequences of SEQ ID NOs: 5 to 8 becomes a worsening factor.
  • FIG. 1 shows the structure of PGSF1.
  • the upper represents the PGSF1 genome, and the lower represents the cDNA structure of PGSF1.
  • the tall and short boxes in the cDNA structure represent the protein coding and noncoding regions, respectively. Closed squares indicate regions conserved in the soybean nodulin family (see Figure 4).
  • AW583046 differs in splice at Exon 2 and has a 53 bp insert that results in a frameshift in the pituitary ORF.
  • FIG. 2 shows an amino acid sequence alignment of PGSF1.
  • FIG. 3 shows multi-tissue Northern hybridization of PGSF1 and actin.
  • FIG. 4 shows an amino acid sequence alignment of PGSF1 and soybean nodulin family. Amino acids corresponding to PGSF1 are shown in black. N ⁇ 2B—S0YBN represents soybean nodulin 26 B, 023 S0YBN represents soybean nodulin 23, and N04 SOYBN represents soybean nodulin 44.
  • FIG. 5 shows the structures of PGSF2 and IGDC1.
  • the tall squares and the short squares represent the protein coding and non-coding regions, respectively. Shaded cells indicate different nucleotide sequences.
  • the signal peptide, the Ig-like region, the transmembrane region (TMs), and the probe used in Northern hybridization are shown.
  • FIG. 6 shows the amino acid sequence alignment of PGSF2I GDC1 and InhBP-s. Matching amino acids are shown in black.
  • FIG. 7 shows multi-tissue northern hybridization using 3 and 5′-probes of PGSF2.
  • FIG. 8 shows multi-tissue northern hybridization of KIAA0512.
  • FIG. 9 shows the inhibitory effect of recombinant human PGSFla on positive samples.
  • FIG. 10 shows the inhibitory effect of recombinant human PGSFla on positive and negative samples.
  • the gene found to be specifically expressed in human pituitary gland found in the present invention can be used as follows.
  • the exon structure of each gene can be determined. Based on this, a primer is prepared to amplify the protein coding region by PCR, and DNA extracted from patient blood is amplified by PCR, the base sequence is determined, and the difference from the normal sequence is examined. That is, primers for PCR-amplifying any of the protein coding regions of SEQ ID NO: 14 are useful for such diagnosis.
  • the nucleotide sequence can be determined by PCR amplification using the following three sets of primers.
  • Tissues such as pituitary tumors are determined by examining gene expression using the quantitative and qualitative methods such as Northern method, PCR method, DNA tip, etc. using the partial sequence of the cDNA of SEQ ID NOs: 1-4. Diagnosis of gene expression. That is, the partial sequence of the cDNA of SEQ ID NOs: 1 to 4 can be used as a diagnostic probe for such diagnosis.
  • Example 2 Using an antigen consisting of the protein having the sequence of SEQ ID NOs: 5 to 8, the autoantibodies against it are measured to determine the autoimmune diseases such as autoimmune pituititis, rheumatoid arthritis, and tissue destruction such as the pituitary gland. Used for diagnosis.
  • a method of reacting a patient's serum with a labeled antigen (each of the novel proteins) to form a labeled antigen / autoantibody complex and then detecting this complex (radioligand assay) is described. Shown.
  • an antibody against the protein having the sequence of SEQ ID NOS: 5 to 8 is prepared, and the protein content of each new protein in serum and tissue is measured by the immunoassay.
  • SEQ ID Nos. 5 to 8 By administering proteins of any sequence or DNAs encoding these proteins (SEQ ID NOS: 1-4), low expression of these proteins can be exacerbating factors, including pituitary diseases. To treat diseases with abnormal endocrine secretions and rheumatoid arthritis.
  • Example 2 In a disease in which low expression of a novel protein (pi-a, PGSFla, PGSFlb, PGSF2) is an exacerbating factor, replacement or treatment is performed by intravenous or oral administration of the novel protein.
  • a novel protein pi-a, PGSFla, PGSFlb, PGSF2
  • Example 3 New tanno II.
  • Low pituitary expression of proteins (pi-a, PGSFla, PGSFlb, PGSF2) is a negative factor, and expression vectors containing DNAs encoding these proteins (SEQ ID NOS: 1 to 4) Replacement and treatment by administration to the pituitary gland.
  • Overexpression of a protein of any one of SEQ ID NOs: 5 to 8 is a disease of exacerbation factor.
  • Antibodies against these proteins or antisense nucleic acids are administered to neutralize the effect and pituitary gland. Treat diseases such as diseases with endocrine and exocrine abnormalities and chronic rheumatoid arthritis.
  • Anti-PGSFla antibody is injected intra-articularly into rheumatoid arthritis patients to suppress the amount of protein and treat it.
  • Example 2 In a disease in which overexpression of a novel protein (pi-a, PGSFla, PGSFlb, PGSF2) is an exacerbating factor, the amount of the protein is suppressed and treated by intravenous administration of an antibody against the novel protein.
  • a novel protein pi-a, PGSFla, PGSFlb, PGSF2
  • Example 3 A disease in which the overexpression of novel proteins (pi-a, PGSFla, PGSFlb, PGSF2) in the pituitary gland is an exacerbation factor. Antisense nucleic acid is administered to the pituitary gland to suppress the expression and treat it.
  • Example 1 Novel protein (pi-a, PGSFla, PGSFlb, PGSF2) is synthesized in vitro or chemically in various cells such as animal cells, insect cells, yeast, and Escherichia coli, and administered to cultured cells or animals. It is used for research on functional analysis by overexpression experiments.
  • Example 2) An antibody against a novel protein is prepared to obtain an antibody that recognizes the primary structure and tertiary structure of the protein and a neutralizing antibody that neutralizes the activity. Recognition of primary structure and three-dimensional structure Antibodies that we know are useful for studying the structure of protein structures. Neutralizing antibodies suppress protein activity when administered to cultured cells or animals, and are useful for functional analysis studies.
  • Example 3 A protein expressed in the living body or in an expression system where the activity is obtained is purified using the antibody as the indicator of activity and activity, and the sugar chain structure is analyzed using the purified protein, or the solid is analyzed by crystal analysis. Examine the structure.
  • the present invention will be illustrated by way of examples, but these are not intended to limit the present invention.
  • test method used in this example is described below.
  • RNA samples from 21 persons aged 70 years were purchased from CLONTEC (Palo Alto, CA, USA). From these, a three-way cDNA library was constructed, and Escherichia coli was transformed according to the report (Okubo K et al. Nature Genetics 2 173-179 (1992)). Briefly, the procedure was as follows: cDNA was synthesized from the above sample using a pUC19-based vector primer digested with MboI, and it was cyclized and introduced into E. coli. This was spread on a plate, and the cDNA insert of a randomly selected transformant was amplified with PCR to produce sequence type II.
  • GS s The resulting 3 'terminal base sequence (hereinafter referred to as "GS s".) Compared to each other Rere by BLAST method to cluster I spoon (Altschul SF et al. Nature Genetics 6 119-129 (1994)) 0 One representative GS from each cluster was selected and compared to a representative sequence from a previously generated cluster. One for each independent cluster GS number. The representative sequence of this group of GS clusters (GS species) was searched for in the DDBJ / GenBank / EMBL database using the regenerated BLAST method (Altschul SF et al. Nature Genetics 6 119-129 (1994)). Comparison of RNA expression in a computer (in silico)
  • the frequency of each GS was compared with that of the body map database.
  • the specific expression of each GS species in the human pituitary is represented by the “pituitary TS ratio” in the following formula.
  • the human pituitary cDNA library constructed using the Zap-cDNA synthesis kit (STRATAGENE, La Jolla, CA, USA) was screened by a standard method.
  • the probe, labeled with alkaline phosphate for the GS sequence was synthesized by Alphos Graphicsystems (Amersham Pharmacia, Arlington Height, IL, USA).
  • Alphos Graphicsystems Alphos Graphicsystems (Amersham Pharmacia, Arlington Height, IL, USA).
  • the phage plaque was transferred to a nylon membrane (Hybond N +, Amersham Pharmacia) by the usual alkaline transfer method, the membrane was heated at 80 ° C for 2 hours. Next, the membrane was hybridized at 55 ° C. with each probe.
  • Adapter primer 1 and primer for GS9544 (5'-CTCAGACCAC CCCTCCTCCC ACGCA-3 '(SEQ ID NO: 16)
  • primer for GS9589 (5 for TGTCTCTTC CAGTAGCAGC ACCGGTAAA-3'
  • Tokimi IJ PCR was performed using primers (5'-AGACAGACCC TCCAAAGATT CA-3 '(J No. 18)) for GS9573.
  • Primers specific to AP1 or genes for this PCR product The sequence was determined using DNA.
  • RNA from human pituitary and liver was fractionated on formaldehyde-agagarose gel and transferred to Hybond N + membrane by the usual alkaline transfer method.
  • This membrane or the human heart, moon, liver, pancreas, skeletal muscle and lung mRNA containing Northern LIGHT Human Multiple mRNA Blot I (Life Technologies, Gaithersburg, MD, USA) was screened using a cDNA library.
  • the protein coding region of the cDNA of the obtained positive clone was hybridized with a cDNA probe labeled with an ECL direct system (Amersham Pharmacia) and washed. This film was exposed to the RX-U diagnostic film at room temperature for 1 hour or more using a sensitizing film.
  • These cDNA probes were obtained by PCR using the primers shown in Table 1.
  • GS9544 GACAGGACGGCGAAGTTTGA TCCTTATTGCCTCACCATTTCC
  • the inventors constructed a human pituitary gene expression profile as described above. This profile shows the relative activity of the gene in retaining the specificity of the tissue.
  • a GS of 1015 was obtained from the human pituitary 3'-directional cDNA library (Table 2).
  • a is a variant of the GNAS1 gene (see GS1007 in Table 3).
  • HLA-C 4 2.59 135 7 41 12 55 16 NM_002117
  • BM-002 3 14.57 18 5 2 4 2 2 NM_016617 galanin-related peptidi e 3 23.84 11 0 1 0 7 0 NMJ15973 1AA0343 3 29.14 9 6 0 0 0 0 AB002341 IAA0512 3 65.57 4 1 0 0 0 0 NM— 014782 Poddy Map database
  • the GNAS1 gene has three promoters, one of which is a secretory granule protein (NESP55),
  • CHGB chromogranin B
  • CPE and 7B2 secretory granule protein genes
  • the fourth most common gene is the GNAS 1 gene.
  • This gene produces three proteins, Gs—hi, extra-large G protein (XL- ⁇ -s) and NESP 55, transcribed by three different promoters (Kozasa T et al. Proceeding of the National Academy of Sciences of the United States of America 85 2081-2085 (1988); Kehlenbach RH et al. Nature 372 804-809 (1994); Ischia R et al. Journal of Biological Chemistry 27211657-11662 (1997).
  • NE SP55 expression is restricted to neuroendocrine tissue, but activity on G s- ⁇ is detected in various tissues, and conversion between its activity and inactivity is due to abnormal endocrine disorders, such as Albright's genetic bone malformation.
  • Ribosomal protein genes (9.9%) and nucleoprotein genes (4.8%) are frequently expressed. Since the two commonly used standards, i3-actin and glyceraldehyde-3-phosphate dehydrogenase, are not expressed uniformly or frequently in tissues in the Body Map Database, some of these genes are expressed in tissues. It will be a good internal standard when comparing levels.
  • Hu et al. Published the expression profile of the bacterial pituitary gland (Hu RM et al. Proceeding of the National Academy of Sciences of the United States of America 97 9543-9548 (2000)). It is based on 7,000 5'-EST from this protein cDNA, one third of which contains the first ATG. They compared it to the hypothalamic and adrenal expression profiles. Consistent with our observations, GH (growth hormone) and PRL (prolactin) were expressed in more than 1% of all ESTs analyzed.
  • the TSH (thyroid stimulating hormone) i3 cDNA lacks the MboI recognition sequence (Tatsumi K et al. Gene 73 489-497 (1988)) and does not appear as GS.
  • the very high expression of PRL in our profile is not due to specific endocrine conditions. The reason is as follows. Since the human pituitary poly (A) RNA used here is derived from 21 humans, tissues from one or two individuals may contain PRL as a result of either prolactinoma or endocrine conditions. Even with high expression, this large amount of PRL Because of the dilution of more than two-fold, the exceptional individuals expressing very high levels of PRL used in our profile express PRL in more than 100% of the total mRNA, a virtually impossible situation.
  • GS species that are frequently and specifically expressed in the pituitary gland were selected (Table 5). Of the 11 GS species, 8 were known genes. Five of them (PRL, GH, LHJ3, glycoprotein alpha, FSHJ3) are anterior pituitary hormones, two (CHGB, PRKACA) are specific to endocrine tissues, and one ( ⁇ ) In 2 ), the characteristics were not determined.
  • s-pamashita® represents pituitary-specific
  • ppd represents pituitary-specific pituitary-predominant.
  • GS9544 By screening the human pituitary cDNA library with the GS sequence, cDNA clones of GS9544, GS9589 and GS9651 were obtained. Although GS9651 was a unique sequence in the human genome draft sequence (International Human Genome Sequencing Consortium 2001, Venter et al 2001, Nature 409 860-921 (2001)), screening of cDNA clones was unsuccessful. According to Northern hybridization, GS9544 and GS9589 were pituitary-specific, and GS9573 (KIAA0512) was the most prominent of the pituitary. Characterization of a novel pituitary-specific transcript
  • PGSFla 128 amino acids
  • PGSFlb 91 amino acids
  • a single partial human cDNA from the Langerhans Island DNA library represented another splice variant (ACC # AW583046) (FIG. 1).
  • the cDNA probes for the two PGSF1s (PGSFla and PGSFlb) strongly hybridize with about 1 kb of pituitary (Pituitary) transcripts in multi-tissue northern hybridizations, resulting in a size of about 1.4 kb. It weakly hybridized to the pancreas transcript (Fig. 3). These are probably endocrine-specific transcripts that are specifically expressed in the pituitary gland.
  • nodulin is a protein associated with the bacterial membrane of the soybean root nodule, whose carboxy-terminal domain is located in the cytoplasmic portion of the nodule. This domain's machine! Identified and rare Reka S (Panter S et al. Molecular Plant-Microbe Interactions 13 325-333 (2000)), the fact that this domain force is retained between S and D Indicates that it has a new function.
  • the full length cDNA of GS9589 is 2 kb long and is predicted to encode a PGSF2 protein of 242 amino acids.
  • this cDNA was identical to immunoglobulin (Ig) -like domain exons 1-5 containing 0.8 kb force 1 (IGDC1) in 5 (Frattini A et al. Gene 214 1- 6 (1998)) and found to be a human homologous protein to the recently identified short splice product of rat inhibin-binding protein, InhBP-S (Bernard DJ & Woodruff K, Molecular Endocrinoloay 15 654). -667 (2001)) (Figs. 5 and 6).
  • This IGGD1 protein consists of an N-terminal single peptide, 12 Ig-like domains and a C-terminal transmembrane domain.
  • PGSF2 is a splice variant of IGDC1 and is probably secreted.
  • the probe for PGSF2 hybridizes with a transcript unique to the pituitary (Pituitary) of about 2 kb by multi-tissue Northern hybridization (Fig. 7).
  • the 3'-probe hybridizes with pituitary-specific transcripts, while the 5'-probe hybridizes with the 2 kb band encoding PGS F2 and IGDC1, respectively, and longer bands (primary). 7).
  • InhBP-L a long splice product of InhBP, was identified as a homologous protein of IGDC1 and a unique receptor for Inhibin A (Bernard DJ & Woodruff TK, Molecular Endocrinology 15 654-667 (2001)).
  • PGSF2 / InhBP-S and IGDCl / InhBP-L are both specifically detected in human and rat tissues with secretory granules (Frattini A et al. Gene 214 1-6 (1998)). Is expressed.
  • a BLAST search on the draft human genome sequence revealed that PGSF2 was located in the q25-26.2 region of chromosome X.
  • the gene for recessive pituitary dysfunction dwarfism linked to X is present in this region (Lagerstrom-Fermer M et al. American Journal of Human Genetics 60 910-916 (1997)). If PGSF2 is associated with this disease, it may be related to pituitary development and function.
  • the full-length GS9573 cDNA is identical to the cDNA encoding KIAA0512, except that it has a 44 bp insert in the 5'-noncoding region.
  • KIAA0512 is predicted to encode a protein consisting of 632 amino acids, has an armadillo] 3-force tenin-like repeat motif, and has 61% homology with pituitary protein (ACC # AF211175) within 307 amino acids.
  • Multi-organized Northern Hanoko According to Zession, KIAA0512 was expressed as 3.2 kb and 4.5 kb transcripts in all tissues tested. Although its expression level is different, it was most strongly expressed in pituitary (Pituitary) and heart (Heart) (Fig. 8).
  • This pituitary protein (ACC # AF211175) binds in vivo to human pectin, which is expressed outside of erythrocytes. Since the sctulin family is a structural protein that exists in association with the membrane or Golgi apparatus, the KIAA0512 protein interacts with sctulin or other spectrin-binding proteins (eg, ankyrin, actin or band 4.1). It may form a complex and may be involved in the transport of parcels.
  • the radioligand assay was used to examine the autoantibody titer to the pituitary-specific protein PGSFla present in the serum of patients with rheumatoid arthritis.
  • rheumatoid factor which detects antibodies to denatured IgG, has been mainly used, but rheumatoid factor has a low positive rate in the early stages of rheumatoid arthritis.
  • an RLA was developed that can be used as an antigen while maintaining its three-dimensional structure if cDNA is present, and that it can also detect three-dimensional structure-recognizing antibodies. was done. Table 6 shows the patient samples used in this example.
  • Serum 4 [mu] 1 was diluted with Reaction buffer (as above) 26 ⁇ 1 35 S - 4 ° C De ⁇ reaction mixed with GH 20 ⁇ 1.
  • Protein G Sepharose (Amersham) 4. Then, the cells were blocked with a blocking buffer (same as above) for 1 hour. 6.4 The “Serum I 35 S-PGSFla” mixture was transferred to a blocked 96-well filter plate, and 10 ⁇ l of Protein G Sepharose (5) was mixed and reacted at room temperature for 45 minutes.
  • PGSFla Recombinant PGSFla was prepared as follows. That is, PGSFla with His tag was expressed in a recombinant protein poor in Escherichia coli (pET expression system, Novaaen) and purified with an affinity column of ALON Metal Affinity Resins (CLONTECH).
  • the suppression test was performed as follows. At the time of dilution of the positive sample, the recombinant PGSFla or ovalbumin (negative control) was reacted with calo;
  • the results are shown below.
  • Table 7 shows the frequency of anti-PGSFla antibody positive in serum.
  • anti-PGSFla antibodies were detected in the serum of patients with rheumatoid arthritis at a frequency of about 80%. Not found in 36 healthy subjects, 20.0% for related osteoarthritis, 0 to 12.5% for autoimmune disease, significantly lower in positivity, especially high in RF-negative patients with rheumatoid arthritis Therefore, it is considered to be effective for supplementary diagnosis of patients with rheumatoid arthritis, which is equal to or better than the measurement of RF.

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Abstract

L'invention concerne des gènes exprimés de manière spécifique dans l'hypophyse, ainsi que des acides aminés. Des études portant sur des gènes exprimés activement dans l'hypophyse humaine ont permis de spécifier des gènes fortement exprimés dans les tissus de l'hypophyse humaine. On a découvert que les gènes de SEQ ID NOS: 1 à 4 sont exprimés de manière spécifique dans l'hypophyse humaine. Sur la base de ces découvertes, on a établi des méthodes permettant d'utiliser efficacement ces gènes et ces protéines. En d'autres termes, l'invention se rapporte à l'utilisation d'une séquence de base représentée par SEQ ID NOS: 1 à 4 ou d'un fragment correspondant dans des examens relatifs à l'hypophyse humaine ou des traitements de maladies associées à l'hypophyse humaine.
PCT/JP2003/002109 2002-02-27 2003-02-26 Methode d'utilisation de genes specifiques a l'hypophyse WO2003072779A1 (fr)

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EP2336769A1 (fr) * 2009-12-18 2011-06-22 F. Hoffmann-La Roche AG Test pour différencier les troubles rhumatismaux des troubles non rhumatismaux
EP4296671A1 (fr) * 2021-02-17 2023-12-27 Fujita Academy Marqueur d'adénohypophysite lymphocytaire et de maladies associées, et utilisation dudit marqueur

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WO1997024436A2 (fr) * 1995-12-28 1997-07-10 Takeda Chemical Industries, Ltd. Polypeptides formant liganos vis-a-vis de la proteine receptrice liee a la proteine g-, leur fabrication et utilisation
WO1998030576A1 (fr) * 1996-10-07 1998-07-16 The Johns Hopkins University School Of Medicine Nouveaux polypeptides derives d'une proteine herisson
WO2000036104A1 (fr) * 1998-12-16 2000-06-22 Zymogenetics, Inc. Polypeptide pancreatique zsig66
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WO2000058473A2 (fr) * 1999-03-31 2000-10-05 Curagen Corporation Acides nucleiques comprenant des phases de lecture ouverte codant des polypeptides; «orfx»
WO2001031003A1 (fr) * 1999-10-29 2001-05-03 Pierre Fabre Medicament Clonage, expression et caracterisation d'un gene exprime dans des cellules tumorales et implique dans la regulation de la reponse immune
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