[go: up one dir, main page]

WO2000020575A1 - Gene associe a la pollinose - Google Patents

Gene associe a la pollinose Download PDF

Info

Publication number
WO2000020575A1
WO2000020575A1 PCT/JP1999/005506 JP9905506W WO0020575A1 WO 2000020575 A1 WO2000020575 A1 WO 2000020575A1 JP 9905506 W JP9905506 W JP 9905506W WO 0020575 A1 WO0020575 A1 WO 0020575A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
hay fever
preparing
compound
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP1999/005506
Other languages
English (en)
Japanese (ja)
Inventor
Takeshi Nagasu
Yuji Sugita
Tomoko Kashiwabara
Tadahiro Oshida
Masaya Obayashi
Shigemichi Gunji
Izumi Obayashi
Yukiho Imai
Ning Lu
Kaoru Ogawa
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genox Research Inc
Original Assignee
Genox Research Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genox Research Inc filed Critical Genox Research Inc
Publication of WO2000020575A1 publication Critical patent/WO2000020575A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the present invention relates to a gene associated with hay fever, a method for examining hay fever using the expression of the gene as an index, and a method for screening a candidate compound for a therapeutic agent for hay fever.
  • Allergic diseases including hay fever, are considered to be multifactorial diseases. These diseases are caused by the interaction of the expression of many different genes, and the expression of these individual genes is affected by several environmental factors. Therefore, it is very difficult to elucidate the specific genes that cause specific diseases.
  • allergic diseases are thought to be related to the expression of genes having mutations or defects, or overexpression or reduced expression of specific genes.
  • genes are involved in pathogenesis and how external stimuli, such as drugs, alter gene expression.
  • the differential display (DD) method is useful as such a method.
  • the differential display method was first developed by Liang and Pardee in 1992 (Science, 1992, 257: 967-971). By using this method, it is possible to screen dozens or more types of samples at one time, and it is possible to detect genes whose expression has changed in those samples. Using such a method, Examining the genes that occur and those whose expression changes with time and environment is expected to provide important information for the elucidation of pathogenic genes. These genes include those whose expression is affected by environmental factors.
  • hay fever is one of the diseases seen in many people in recent years.
  • the etiology of hay fever may be related to multiple genes whose expression is affected by pollen, one of the environmental factors. Under such circumstances, it has been desired to isolate a gene associated with hay fever. Disclosure of the invention
  • An object of the present invention is to provide a gene associated with an allergic disease, particularly, hay fever. Furthermore, another object of the present invention is to provide a method for examining hay fever and a method for screening a candidate compound for a therapeutic agent for hay fever using the expression of the gene as an index.
  • the present inventors prepared from a plurality of human blood based on the procedure of the already established “Fluorescent DD (Fluorescent DD) method” (T. Ito et al., 1994, FEBS Lett. 351: 231-236).
  • Fluorescent DD Fluorescent DD
  • the present inventors collected T cells from blood before and after pollen dispersal for a plurality of subjects including pollinosis patients, and between subjects with different cedar pollen-specific IgE values and before and after pollen dispersal.
  • the present inventors divided the subjects into a group with a high IgE value for cedar pollen (predisposed to cedar pollinosis) and a group other than those (healthy subjects), and determined the expression level of the isolated “B759” gene in both groups. As a result of comparative analysis, it was found that the gene showed a significantly higher value in the cedar pollinosis-diseased group than in healthy subjects. Therefore, the present inventors have conducted a hay fever test and a screening of a candidate compound for a therapeutic drug for hay fever using the expression level of the gene as an index. It was found that it could be done.
  • the present invention relates to a gene that is highly expressed in a person predisposed to hay fever, a method for testing hay fever using the expression of the gene as an index, and a method for screening a candidate compound for a therapeutic agent for hay fever.
  • a gene that is highly expressed in a person predisposed to hay fever a method for testing hay fever using the expression of the gene as an index, and a method for screening a candidate compound for a therapeutic agent for hay fever.
  • nucleic acid molecule comprising the base sequence of SEQ ID NO: 1 or a partial sequence thereof
  • nucleic acid molecule comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1,
  • the polymerase chain reaction is performed by a PCR amplification monitor method, as described in (6). the method of,
  • a method for screening a therapeutic compound candidate compound for hay fever comprising:
  • step (f) selecting a compound that reduces the amount of RNA measured in step (e) as compared to a control (in the case where no test compound is administered),
  • a method for screening a candidate compound for a therapeutic agent for hay fever comprising:
  • step (f) selecting a compound that reduces the amount of DNA amplified in step (e) compared to a control (in the case where the test compound is not administered), (12) A method for screening a therapeutic compound candidate for hay fever, comprising:
  • step (h) selecting a compound that reduces the amount of UNA measured in step (g) as compared to a control (when no test compound is administered);
  • a method for screening a candidate drug for treating hay fever comprising:
  • step (h) selecting a compound that reduces the amount of DNA amplified in step (g) as compared to a control (in the case where no test compound is administered);
  • a method for screening a candidate drug for treating hay fever comprising: (a) preparing lymphocytes from a hay fever model animal or human having hay fever; (b) stimulating the lymphocytes with a pollen antigen in the presence of a test compound,
  • step (g) selecting a compound that reduces the amount of RNA measured in step (f) compared to a control (in the case where the test compound is not administered),
  • a method for screening a therapeutic compound candidate for hay fever comprising:
  • step (g) selecting a compound that reduces the amount of DNA amplified in step (f) as compared to a control (in the case where the test compound is not administered);
  • a method for screening a candidate compound for treating a hay fever comprising:
  • step (e) selecting a compound that reduces the amount of RNA measured in step (d) as compared to a control (in the case where the test compound is not administered),
  • a method for screening a candidate compound for treating a hay fever comprising:
  • step (e) selecting a compound that reduces the amount of DNA amplified in step (d) as compared to a control (in the case where the test compound is not administered),
  • lymphocytes are prepared from peripheral blood
  • the “nucleic acid molecule” in the present invention includes DNA and RNA.
  • the “test for hay fever” in the present invention includes not only a test for a patient who has developed hay fever, but also a determination of whether or not a subject who has not developed hay fever has a predisposition to hay fever. Inspections are also included.
  • the present invention relates to a novel gene “B759” that is correlated with an IgE production response to cedar pollen of an individual.
  • the nucleotide sequence of the “B759” cDNA found by the present inventors is shown in SEQ ID NO: 1.
  • B759 refers to the atopy predisposing group (for cedar pollen before and after pollen scattering). IgE level of 3.5 AU / ml or more) showed higher expression than the non-atopic group. Therefore, it would be possible to conduct hay fever testing and screening for hay fever therapeutic drug candidates using the expression of B759 gene (including transcription into mRNA and translation into protein) as an index. .
  • cedar pollinosis is particularly preferred as a hay fever as a control for examination and treatment.
  • the detection of the expression of the "B759" gene in the examination of hay fever according to the present invention can be performed by a hybridization technique using a nucleic acid that hybridizes to the "B759" gene as a probe, or a primer that hybridizes to the DNA of the present invention. It is possible to use the gene amplification technology described above.
  • nucleic acid molecule that specifically hybridizes to the “B759” gene and has a chain length of at least 15 nucleotides is used.
  • the term “specifically hybridizes” as used herein refers to a DNA that cross-links with another gene encoding DNA and / or RNA under ordinary hybridization conditions, preferably under stringent hybridization conditions. It means that the dimensioning does not occur significantly.
  • These nucleic acid molecules may be synthetic or natural.
  • probe DNA used for hybridization is usually labeled.
  • Labels include, for example, labeling by nick translation using DNA polymerase 1, end labeling using polynucleotide kinase, and filin end labeling using Klenow fragment (Berger SL, Ki t el AR. (1987) Guide to Molecular Cloning Techniques, Method in Enzymology, Academic Press; Hames BD, Higgins SJ (1985) Genes Probes: A Practical Approach.IRL Press; Sambrook J, Fritsch EF, Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edn.
  • RNA polymerase Melt DA, Krieg, PA, Rebagkiti MR, Maniatis T, Zinn K, Green MR. (1984) Nucleic Acid Res., 12, 7035-7056
  • the method of incorporating the modified nucleotide that does not use radioactive isotopes for DNA testing (KrickaLJ. (1992) Nonisotopic DNA Probing Techniques. Academic Press) and the like pollinosis using c High Priestess die internalization techniques include, for example, Roh one
  • the method can be carried out by using a hybridization method, a dot blot method, a method using a DNA microarray, or the like.
  • an RT-PCR method can be used as a method using the gene amplification technique.
  • the expression of the “B759” gene can be more accurately quantified by using the PCR amplification monitor method as shown in Example 8 in the gene amplification process.
  • probes that are labeled with different fluorescent dyes that cancel each other's fluorescence at both ends are used to hybridize to the detection target (reverse transcript of DNA or RNA).
  • the detection target reverse transcript of DNA or RNA.
  • the two fluorescent dyes separate and the fluorescence is detected. This fluorescence is detected in real time.
  • the number of copies of the target in the target sample is determined by the number of linear cycles of PCR amplification by simultaneously measuring a standard sample with a clear copy number for the target (Holland, PM et al., 1991, Proc. Natl. Acad. Sci.
  • the hay fever test of the present invention may be performed by detecting the protein encoded by “B759”.
  • an inspection method for example,
  • a Western blotting method, an immunoprecipitation method, an ELISA method and the like using an antibody that binds to the protein encoded by “B759” can be used.
  • the antibody of the protein encoded by "B759” of the present invention can be prepared by techniques known to those skilled in the art. It can be obtained as a polyclonal antibody or a monoclonal antibody using the method (Milstein C, et al., 1983, Nature 305 (5934): 537-40).
  • the protein or its partial peptide used as an antigen can be obtained by, for example, inserting the “B759” gene or a part thereof into an expression vector, introducing this into an appropriate host cell, producing a transformant, and transforming the transformant. It can be obtained by culturing to express the recombinant protein, and purifying the expressed recombinant protein from a culture or a culture supernatant.
  • the expression of the gene of the present invention is significant. If it is too high, the subject has a high IgE value for cedar pollen and can be determined to have a predisposition to hay fever.
  • the measurement of the expression level of the gene of the present invention, together with the pollen-specific antibody titer, symptoms, etc., can be used for hay fever testing.
  • the screening method for a candidate compound for treating hay fever of the present invention can be performed in vivo or in vitro.
  • in vivo screening for example, after administering a candidate drug and stimulating with a pollen antigen to a model animal such as a mouse, T cells are separated from peripheral blood, and the transcript of “B759” is measured. .
  • lymphocytes are separated from peripheral blood, and the lymphocytes are stimulated in vitro with cedar pollen antigen or the like. T cells are separated from the lymphocytes after the stimulation, and the transcript of the “B759” gene is measured.
  • a compound that suppresses the transcription level of the “B759” gene is selected.
  • peripheral blood lymphocytes are collected from humans or mice, and the peripheral blood lymphocytes are stimulated in vitro with cedar pollen antigen or the like.
  • Candidate compounds are added during in vitro stimulation.
  • T cells are separated from the stimulated peripheral blood lymphocytes, and the transcript of “B759” is measured. As a result of this measurement, a compound that suppresses the transcription level of the “B759” gene is selected.
  • screening for a candidate compound for treating hay fever of the present invention can also be performed using established T cells.
  • established T cells such as Molt4 cells and Jurkat cells are stimulated in vitro with lymphocyte stimulants.
  • Lymphocyte stimulants include, for example, cal Examples include simmionophore (A23187), PMA, and phytohemagglutinin (PHA).
  • Add candidate drugs during in vitro stimulation Thereafter, the transcription amount of the “B759” gene in the established T cells is measured. As a result of this measurement, a compound that suppresses the transcription of the “B759” gene is selected.
  • Detection of the expression of the "B759" gene in screening for a candidate compound for the treatment of hay fever can be performed by the hybridization technique using a nucleic acid that hybridizes to the "B759" gene as a probe, as in the examination of the hay fever of the present invention, or by using It can be carried out using a gene amplification technique using a DNA that hybridizes to the gene of the present invention as a primer.
  • a method using the hybridization technology for example, a Northern hybridization method, a dot blot method, a method using a DNA microarray, or the like can be used.
  • an RT-PCR method can be used as a method utilizing gene amplification technology. In the RT-PCR method, more accurate quantification of the expression of the “B759” gene can be performed by using a PCR amplification monitor method as shown in Example 8 in the gene amplification process.
  • test compounds used in these screenings include steroid compounds such as steroid derivatives, compound compounds synthesized by existing chemical methods, compound compounds synthesized by combinatorial chemistry, and extracts of animal and plant tissues. Alternatively, a mixture containing a plurality of compounds such as a microorganism culture, and a sample purified therefrom may be mentioned.
  • the compound isolated by the method for screening a candidate compound for a therapeutic agent for pollinosis of the present invention is a candidate for a drug which improves allergic predisposition to pollen.
  • the compound isolated by the screening method of the present invention when used as a pharmaceutical, it can be used as a pharmaceutical preparation by a known pharmaceutical production method.
  • a pharmaceutically acceptable carrier or vehicle such as saline, vegetable oils, suspensions, surfactants, stabilizers, etc.
  • Administration will be transdermal, intranasal, transbronchial, intramuscular, intravenous, or oral, depending on the nature of the compound.
  • the dose varies depending on the patient's age, body weight, symptoms, administration method and the like, but those skilled in the art can appropriately select an appropriate dose.
  • FIG. 1 is a diagram showing the antibody titers of cedar pollen-specific IgE antibodies in a total of 18 blood samples of 10 subjects who collected blood.
  • the value of cedar pollen-specific IgE antibody in each blood sample of subjects A to J was expressed in AU / ml. Before pollen scattering is shown on the left (white column) and after pollen on the right (black column). Subjects A and B collected only blood after pollen scattering.
  • FIG. 2 is a graph showing changes in the expression of “B759” in a high IgE group and a normal IgE group when classified according to cedar pollen-specific IgE values.
  • the shaded area indicates the high IgE group, and the white area indicates the normal IgE group. Error bars represent standard deviation.
  • Fig. 1 shows the measured cedar pollen-specific IgE values before and after pollen scattering in each subject. As shown, most of the 10 subjects had increased serum levels of cedar pollen-specific IgE after pollen exposure. Whether or not they had a predisposition to atopy was judged based on whether they were greater than the value of the CAP RAST test for cedar pollen-specific IgE. That is, eight subjects A to G and I were regarded as atopic predisposition group (hereinafter also referred to as “patient”), and two subjects H and J were regarded as healthy subjects (hereinafter also referred to as “normal group”). Of the eight atopic subjects, seven exhibited symptoms of allergic rhinitis after pollen dispersal.
  • patient atopic predisposition group
  • normal group healthy subjects
  • the procedure was as follows. First, the wall of the syringe was uniformly treated with 1 ml of heparin from Novo, etc., and blood was collected in a 10 ml syringe containing heparin at a final concentration of 50 unit / ml. At this time, two 22G needles were prepared for one blood sample. The needle was removed and transferred to a 50 ml centrifuge tube (made of polypropylene).
  • PRP Plate rich plasma, platelet-rich plasma
  • PRP Platelet rich plasma, platelet-rich plasma
  • the precipitated cells were suspended in 5 ml of Ca- and Mg-free HBSS obtained from Gibco or the like. This was overlaid on one tube (Falcon tube: 2006 or 2059; made of polypropylene) containing 5 ml of Ficol Paque (Pharmacia) using a pasteur pipet.
  • the tube was centrifuged at 1500 rpm (equivalent to 400 ⁇ g in a centrifuge manufactured by Tomy) for 30 minutes at room temperature.
  • 1500 rpm Equivalent to 400 ⁇ g in a centrifuge manufactured by Tomy
  • granulocytes, Red blood cells (erythrocytes) precipitate and lymphocytes in the middle layer with the ficoll layer in between
  • lymphocyte monocytes, platelets, platelets
  • the lymphocyte fraction obtained in Example 2 was centrifuged at 1200 rpm at 4 ° C. for 5 minutes, and suspended in BSA / PBS at 10 8 per 100 ° C. The capacity became about 20 ⁇ 1. This was transferred to an Eppendorf tube (1.5 ml), and the CD3 microbead solution was added. Then, it was left at 4-10 ° C for 30 minutes (it was not placed on ice at this time). This sample was treated with Magnetic Celso Ichiichi (MACS) (manufactured by Miltenyi Biotech Inc.) as follows.
  • MCS Magnetic Celso Ichiichi
  • the MS + / RS + column was mounted on a Mini MACS or Vario MACS separation unit (without needles). 500 ⁇ 1 of BSA / PBS was gently applied to the column, and the buffer was poured out. Next, the c column in which cells labeled with CD3 microbeads were applied to the column was washed three times at 500 ⁇ 1 (B cell fraction). The column was removed from the separation unit and placed on a tube for collecting the eluate. 1 ml of BSA / PBS was applied to the column, and positive cells were rapidly flushed out using a plunger attached to the column. This was used as the T cell fraction.
  • the obtained T cell fraction was centrifuged at 1200 rpm for 5 minutes at 4 ° C.
  • the precipitate was washed twice with BSA / PBS. After the second washing, the cells were suspended in 1 ml, and a part thereof was diluted 2-fold with a trypan, and the number of cells was counted. Total cell number was approximately 4 ⁇ 10 6 .
  • the cell lysate was stored frozen, it was incubated at 37 ° C for 10-15 minutes, and if insolubles were visible, it was centrifuged at maximum speed for 3 minutes, and only the supernatant was collected.
  • the lysate was homogenized with a syringe equipped with a 20 G force teran needle and then treated with QIAshredder. (That is, usually, a lysate of 350-1 cells was applied to a Kyashredunit using a Bitman. This was centrifuged at 1500 rpm for 2 minutes, and the effluent was collected.) % Ethanol was added and mixed well by pipetting.
  • the RNeasy spin column was attached to the attached 2 ml tube, the cell lysate mixture was applied, centrifuged at 8000 xg (11500 rpm) for 1 minute, and the effluent was discarded. Powshbaffe RW1 700 1 was applied to the column, and the lid was set up for 5 minutes. The mixture was centrifuged at 11,500 rpm for 15 seconds, and the effluent was discarded. The column was mounted on a new 2 ml tube, 500 ⁇ 1 of Wash Buffer RPE (including ethanol) was applied to the column, and the mixture was centrifuged at 11,500 rpm for 15 seconds, and the effluent was discarded.
  • Wash Buffer RPE including ethanol
  • Wash buffer RPE 500 ⁇ 1 was applied to the column and centrifuged at maximum speed for 2 minutes.
  • the column was mounted in a new 1.5 ml tube, 30 ⁇ 1 of DEPC-treated water was applied, the lid was closed, and the column was allowed to stand for 10 minutes. After centrifugation at 11,500 rpm for 10 minutes, total RNA was obtained. Measure the concentration, and if the volume is low, reattach the column to a new 1.5 ml tube, apply 30 ⁇ 1 of DEPC-treated water, close the lid, stand for 10 minutes, and centrifuge at 11500 rpm for 10 minutes did.
  • DNase treatment was performed to remove DNA from total RNA prepared from T cells.
  • DD Fluorescent Differential Display
  • the PCR reaction conditions were as follows: 1 cycle of ⁇ 95 ° C for 3 minutes, 40 ° C for 5 minutes, 72 ° C for 5 minutes '', followed by 30 cycles of ⁇ 94 ° C for 15 seconds, 40 ° C for 2 minutes, and 72 ° C for 1 minute '' Thereafter, the temperature was raised to 72 ° C for 5 minutes, and then continuously to 4 ° C.
  • the primer pairs used were arbitrary primers for the anchor-primer GT15A (SEQ ID NO: 2), GT15C (SEQ ID NO: 3), and GT15G (SEQ ID NO: 4), respectively AG 1-110, AG 111 199 and AG 200-287 were combined for a total of 287 reactions.
  • an oligomer composed of 10 nucleotides having a GC content of 50% was designed, synthesized, and used.
  • a 6% denaturing polyacrylamide gel was prepared, a 2.5 ⁇ 1 sample was applied, and electrophoresed at 40 W for 210 minutes. Then, the gel plate was scanned using Hitachi Fluorescence Image Analyzer-FMB10II, and electrophoretic images were obtained by fluorescence detection.
  • Example ⁇ Amplification and sequencing of band excised by DD analysis Two DD analyzes were performed using a number of arbitrary primers. Bands that differed before and after pollen dispersal or between the patient and healthy groups were selected and reproducible bands were excised from the gel in two experiments.
  • B759 Further analysis was performed on one of the excised bands (referred to as "B759”).
  • the band of “B759” was found by DD analysis using GT15A (SEQ ID NO: 2) as an anchor primer and AG69 (SEQ ID NO: 5) as an arbitrary primer, and the expression of “B759” was 4/8. In humans, the expression was higher before scattering than after scattering.
  • a gel containing the “B759” band was cut out, stored in a TE solution, and heated at 60 ° C. for 10 minutes to elute DNA from the gel.
  • PCR was performed under the same conditions as DD-PCR, and a DNA fragment of about 210 bp was amplified.
  • GT15A was used as an anchor primer, and AG69 was used as an optional primer.
  • the amplified DNA fragment was cloned with a plasmid vector pCR2.1 (Invitrogen) to obtain a plasmid P759-11 having a DNA fragment of about 210 bp.
  • the nucleotide sequence of the DNA fragment was determined using the plasmid DNA according to a conventional method.
  • This method uses a fluorescent dye to quantitatively detect the PCR-amplified DNA strand in real time.
  • Primers 759-5 '(SEQ ID NO: 6), 759-3, (SEQ ID NO: 7), and TaqMan probe 759 SEQS5 (SEQ ID NO: 8) based on the base sequence of the DD band determined in Example 7 was designed, synthesized, and used for quantitative reactions.
  • TaqMan probe 759SEQS5 was used with its 5 'end fluorescently labeled with FAM (6-carboxyfluorescein) and its 3' end fluorescently labeled with TAMRA (6-carboxy-tetramethyl-rhodamine).
  • FAM 6-carboxyfluorescein
  • TAMRA 6-carboxy-tetramethyl-rhodamine
  • reverse transcribed cDNA was used as a primer with poly T (12 to 18) from all 44 types of A.
  • Reaction composition of ABI-PRISM 7700 (reaction amount per liter) Sterile distilled water 25.66 ( ⁇ L)
  • Two-way analysis of variance was performed using the values of. Grouping before and after pollen scattering, and Was divided into two factors: 10 (high IgE group) and 12 (normal IgE group) who showed 3.5 AU / ml or more in at least one measurement of each specific IgE in serum. And tested. The test was performed separately for the group showing 200 AU / ml for total IgE and the other groups. Tests for two-way analysis of variance were performed using StatView software (Abacuus Concepts, Inc.).
  • RH method Radiation Hybrids to determine the location of “B759” on the human chromosome Chromosome mapping was performed using the chromosome method (RH method).
  • the RH method is based on the combination of hybrid cells (RH panel) obtained by irradiating human diploid cells with X-rays to physically cut chromosomes and then fusing them with Hamus Yuichi cells. This is a method in which the position of the gene of interest is determined by examining the presence of the gene of interest by PCR and linkage analysis with about 10,000 known markers (Hudson TJ et al., 1995, Science 270: 1945- 1954 Schuler GD et al., 1996, Science 274: 540-546; Stewart, EA and D.
  • “B759” was mapped to about 2.4 Mb from the D7S649 (Genbank accession number Z23771), which is a marker present on chromosome 7 q32.
  • a peripheral blood T cell cDNA library was prepared using pBluescript II vector (Stratagene) and TimeSaver cDNA Synthesis kit (Pharmacia Biotec). Screening by PCR was carried out using the specific primer 759-11.2. (SEQ ID NO: 10) prepared from the band sequence and the T7 primer (SEQ ID NO: 9) on the pBluescript Vector. This operation was named VD-PCR for convenience. After 94 ° C for 3 minutes, 35 cycles of “94 ° C for 30 seconds, 55 ° C for 30 seconds, and 68 ° C for 2 minutes” were performed, followed by treatment at 68 ° C for 15 minutes. As a result, an amplification product of about 2.2 kb was obtained. C When this MA sequence was determined, it certainly contained the sequence of “B759” determined so far.
  • a novel gene having a correlation with a cedar pollen-specific IgE value was provided.
  • the expression of the gene of the present invention as an index it has become possible to carry out a test to determine whether or not the subject has a hay fever predisposition and to screen a candidate compound for a therapeutic drug for hay fever.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Pulmonology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un nouveau gène ayant une expression sensiblement élevée chez les sujets présentant des taux élevés d'IgE spécifique au pollen de cèdre, isolé avec succès par la préparation de lymphocytes T de sujets présentant différents taux d'IgE spécifique au pollen de cèdre, avant et après la dispersion de pollens et la recherche d'un gène selon la technique du DD. Ledit gène est utilisable dans le dosage de la pollinose et le criblage de composés candidats pour des remèdes contre la pollinose.
PCT/JP1999/005506 1998-10-06 1999-10-06 Gene associe a la pollinose Ceased WO2000020575A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP10284610A JP2000106879A (ja) 1998-10-06 1998-10-06 花粉症関連遺伝子
JP10/284610 1998-10-06

Publications (1)

Publication Number Publication Date
WO2000020575A1 true WO2000020575A1 (fr) 2000-04-13

Family

ID=17680697

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1999/005506 Ceased WO2000020575A1 (fr) 1998-10-06 1999-10-06 Gene associe a la pollinose

Country Status (2)

Country Link
JP (1) JP2000106879A (fr)
WO (1) WO2000020575A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000073435A1 (fr) * 1999-05-27 2000-12-07 Genox Research, Inc. Gene 441 associe a la pollinose
WO2000073439A1 (fr) * 1999-05-27 2000-12-07 Genox Research, Inc. Gene 465 associe a la pollinose
WO2001073022A1 (fr) * 2000-03-29 2001-10-04 Kyowa Hakko Kogyo Co., Ltd. Gene associe a la glomerulonephrite proliferative
WO2002024903A1 (fr) * 2000-09-25 2002-03-28 Genox Research, Inc. Methode d'examen applicable en cas de maladie allergique
WO2002026962A1 (fr) * 2000-09-26 2002-04-04 Genox Research, Inc. Procede d'examen de maladies allergiques

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002277456A (ja) * 2001-03-21 2002-09-25 Jenokkusu Soyaku Kenkyusho:Kk アレルギー性疾患の検査方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06197768A (ja) * 1993-01-07 1994-07-19 Meiji Seika Kaisha Ltd スギ花粉抗原およびそれをコードする遺伝子
JPH0847392A (ja) * 1993-11-05 1996-02-20 Meiji Milk Prod Co Ltd スギ花粉アレルゲンＣｒｙ ｊ ＩＩエピトープ

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06197768A (ja) * 1993-01-07 1994-07-19 Meiji Seika Kaisha Ltd スギ花粉抗原およびそれをコードする遺伝子
JPH0847392A (ja) * 1993-11-05 1996-02-20 Meiji Milk Prod Co Ltd スギ花粉アレルゲンＣｒｙ ｊ ＩＩエピトープ

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KOMIYAMA N. ET AL.: "cDNA cloning and expression of Cry j II, the second major allergen of Japanese cedar pollen", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 201, no. 2, 1994, pages 1021 - 1028, XP002925249 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000073435A1 (fr) * 1999-05-27 2000-12-07 Genox Research, Inc. Gene 441 associe a la pollinose
WO2000073439A1 (fr) * 1999-05-27 2000-12-07 Genox Research, Inc. Gene 465 associe a la pollinose
WO2001073022A1 (fr) * 2000-03-29 2001-10-04 Kyowa Hakko Kogyo Co., Ltd. Gene associe a la glomerulonephrite proliferative
WO2002024903A1 (fr) * 2000-09-25 2002-03-28 Genox Research, Inc. Methode d'examen applicable en cas de maladie allergique
US7148011B2 (en) 2000-09-25 2006-12-12 Japan As Represented By General Director Of Agency Of National Center For Child Health And Development Method of testing for allergic diseases
WO2002026962A1 (fr) * 2000-09-26 2002-04-04 Genox Research, Inc. Procede d'examen de maladies allergiques

Also Published As

Publication number Publication date
JP2000106879A (ja) 2000-04-18

Similar Documents

Publication Publication Date Title
US10017821B2 (en) Biomarkers for diagnosing ischemia
JP6997709B2 (ja) 全身性炎症反応症候群（ｓｉｒｓ）患者における合併症リスクを評価する方法
KR20160080165A (ko) 퇴행성 뇌질환 진단용 조성물 및 이를 이용한 퇴행성 뇌질환의 진단 방법
EP1260816A1 (fr) Methode d'examen de maladies allergiques
WO2000020575A1 (fr) Gene associe a la pollinose
JPWO2002050269A1 (ja) アレルギー性疾患の検査方法
WO2000065046A1 (fr) Gene 373 associe a la pollinose
JP7394441B2 (ja) 脳腫瘍を検査する方法
US6986990B1 (en) Pollen allergy-related gene 513
WO2000073435A1 (fr) Gene 441 associe a la pollinose
WO2000065051A1 (fr) Gene 627 associe a la pollinose
WO2000065050A1 (fr) Gene 795 associe a la pollinose
WO2000073439A1 (fr) Gene 465 associe a la pollinose
JP2003125777A (ja) 花粉症関連遺伝子、５８１
JP2003125776A (ja) 花粉症関連遺伝子、４１９
WO2000073440A1 (fr) Gene 787 associe a la pollinose
JPWO2000073439A1 (ja) 花粉症関連遺伝子、４６５
JPWO2000065051A1 (ja) 花粉症関連遺伝子、６２７
JPWO2001065259A1 (ja) アレルギー性疾患の検査方法
JPWO2000065049A1 (ja) 花粉症関連遺伝子、５１３
JPWO2000065046A1 (ja) 花粉症関連遺伝子、３７３
JPWO2000073435A1 (ja) 花粉症関連遺伝子、４４１
JPWO2000065048A1 (ja) 花粉症関連遺伝子、５８１
JPWO2000065045A1 (ja) 花粉症関連遺伝子、４１９
JPWO2003010310A1 (ja) アレルギー性疾患の検査方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase