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WO2000065046A1 - Gene 373 associe a la pollinose - Google Patents

Gene 373 associe a la pollinose Download PDF

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Publication number
WO2000065046A1
WO2000065046A1 PCT/JP2000/002730 JP0002730W WO0065046A1 WO 2000065046 A1 WO2000065046 A1 WO 2000065046A1 JP 0002730 W JP0002730 W JP 0002730W WO 0065046 A1 WO0065046 A1 WO 0065046A1
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Prior art keywords
cells
preparing
rna
compound
test compound
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English (en)
Japanese (ja)
Inventor
Takeshi Nagasu
Yuji Sugita
Tomoko Fujishima
Tadahiro Oshida
Masaya Obayashi
Shigemichi Gunji
Izumi Obayashi
Yukiho Imai
Nei Yoshida
Kaoru Ogawa
Keiko Matsui
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Genox Research Inc
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Genox Research Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • 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
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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 a gene associated with an allergic disease, in particular, hay fever, a method for testing an allergic disease using an expression of the gene as an index, and a method for screening a candidate therapeutic drug for an allergic disease.
  • Allergic diseases including hay fever, are considered 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 multiple 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 to overexpression or reduced expression of specific genes. To understand the role of gene expression in disease, it is necessary to understand how 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 in 1992 by Liang and Pardee (Science, 1992, 257: 967-971). By using this method, dozens or more of samples can be screened at a time, and It is possible to detect a gene whose expression has changed. Using such a method to examine genes with mutations or genes whose expression changes with time or environment is expected to provide important information for elucidating 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 pathogenesis of hay fever may involve several 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. Another object of the present invention is to provide a method for detecting an allergic disease and a method for screening a candidate compound for a therapeutic drug for allergic diseases, using the expression of the gene as an index.
  • the inventors of the present invention have determined from a plurality of human blood samples based on 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 scattering in multiple subjects including pollinosis patients, and expressed T cells between subjects with different cedar pollen-specific IgE values and before and after pollen scattering.
  • the present inventors divided the subjects into a group having a high IgE value for cedar pollen (a group predisposed to cedar pollinosis) and another group (healthy subjects), and determined the expression level of the isolated “373” gene in both groups. As a result of comparative analysis, it was found that the gene showed a significantly lower value in the cedar pollinosis-diseased group as compared with healthy subjects. Therefore, the present inventors have determined that the expression level of the gene As an index, it has been found that it is possible to conduct an allergic disease test and to screen for a candidate drug for a therapeutic drug for allergic disease.
  • the present invention relates to a gene showing high expression in a person having an allergic predisposition, a method for testing an allergic disease using the expression of the gene as an index, and a method for screening a candidate compound for a therapeutic drug for an allergic disease. More specifically,
  • nucleic acid molecule encoding a protein comprising the amino acid sequence of SEQ ID NO: 2,
  • nucleic acid molecule comprising the coding region of the nucleotide sequence of SEQ ID NO: 1,
  • T cells are prepared from peripheral blood of the subject
  • a method for screening a candidate drug for treating an allergic disease comprising:
  • step (f) selecting a compound that increases the amount of RNA measured in step (e) compared to a control (in the case where the test compound is not administered),
  • step (e) performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to [3] as a primer, (f) selecting a compound that increases the amount of DNA amplified in step (e) compared to a control (in the case where the test compound is not administered),
  • PCR polymerase chain reaction
  • step (h) selecting a compound that increases the amount of RNA measured in step (g) as compared to a control (in the case where the test compound is not administered),
  • a method for screening a candidate drug for treating an allergic disease comprising:
  • step (h) selecting a compound that increases the amount of DNA amplified in step (g) as compared to a control (in the case where the test compound is not administered),
  • step (g) selecting a compound that increases the amount of RNA measured in step (f) 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 an allergic disease comprising: (a a step of preparing lymphocytes from a model animal of hay fever or a human having hay fever,
  • step (g) selecting a compound that increases the amount of DNA amplified in step (f) as compared to a control (in the case where no test compound is administered),
  • a method of screening a candidate compound for a therapeutic agent for an allergic disease comprising: (a) stimulating established T cells with a lymphocyte stimulating substance in the presence of a test compound; Preparing an RNA sample from T cells,
  • step (e) selecting a compound that increases the amount of RNA measured in step (d) compared to a control (in the case where no test compound is administered),
  • step (e) selecting a compound that increases the amount of DNA amplified in step (d) as compared to a control (when no test compound is administered);
  • T cells are prepared from peripheral blood of a hay fever model animal, (10) or the method according to (11),
  • lymphocytes are prepared from peripheral blood
  • [21] a protein comprising the amino acid sequence of SEQ ID NO: 2, and [22] an antibody that binds to the protein of [21].
  • an allergic disease is a general term for diseases associated with allergic reactions. More specifically, it can be defined as identifying the allergen, demonstrating a deep link between exposure to the allergen and the development of the lesion, and demonstrating an immunological mechanism for the lesion.
  • the immunological mechanism means that T cells show an immune response by allergen stimulation.
  • Representative allergies One disease can be bronchial asthma, allergic rhinitis, atopic dermatitis, hay fever, or insect allergy.
  • Allergic predisposition (al lergi c diathes is) is a genetic factor transmitted from parents to children with allergic diseases. Allergic diseases that occur familially are also called atopic diseases, and the genetic factors that cause them are atopic predisposition.
  • the “nucleic acid molecule” in the present invention includes DNA and RNA.
  • the “test for allergic disease” in the present invention includes not only a test for a patient who has an allergic disease, but also a test for determining whether or not a subject who does not have an allergic disease has an allergic predisposition. Inspection is also included.
  • the present invention relates to a novel gene “373” that is correlated with an IgE production response to cedar pollen of an individual.
  • the nucleotide sequence of the "373" cDNA found by the present inventors is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by "373” is shown in SEQ ID NO: 2.
  • the nucleotide sequence of the “373” cDNA isolated by the present inventors is a partial distribution sequence of the “373” cDNA, and those skilled in the art may use the sequence information of the “373” cDNA described in SEQ ID NO: 1.
  • isolation of the full-length cDNA of “373” can be usually performed. That is, a method for screening a T cell cDNA library or the like by hybridization using a sequence derived from “373” as a probe, or a method for screening a T cell cDNA library or the like using a sequence derived from “373” as a primer.
  • a library is screened by using as an indicator that the amplification product of the size specific to the primer is obtained, and the full length of the cDNA is obtained.
  • the RACE method Frohman, MA et al .: Proc.
  • the mRNA derived from T cells is converted to single-stranded cDNA using a sequence derived from “373” as a primer, and oligomers are added to the ends before PCR Natl. Acad. Sc. USA, 85: 8992, 1988).
  • the “nucleic acid molecule encoding a protein containing the amino acid sequence of SEQ ID NO: 2” includes the full-length “373” cDNA isolated in this manner.
  • the total length "3 73 '' Once the cDNA is isolated, insert it into an appropriate expression vector, introduce it into an appropriate host cell, culture the cell, and recover the expressed protein from the cell, A recombinant protein encoded by the cDNA can be prepared.
  • the “protein containing the amino acid sequence of SEQ ID NO: 2” includes the full-length “373” protein thus prepared.
  • “373” showed significantly lower expression in the atopic predisposition group (powder value for cedar pollen was 3.5 AU / ml or more) than in the non-atopic predisposition group. Therefore, it is thought that it is possible to conduct an allergic disease test and a screening of candidate compounds for a therapeutic drug for allergic diseases, using the expression of the “373” gene (including transcription into mRNA and translation into protein) as an index. .
  • cedar pollinosis is particularly preferred as an allergic disease to be tested and treated.
  • Detection of the expression of the “373” gene in the test for allergic disease can be performed by a hybridization technique using a nucleic acid that hybridizes to the “373” gene as a probe, or a DNA that hybridizes to the gene of the present invention. It can be carried out by using gene amplification technology as a primer.
  • a nucleic acid molecule that specifically hybridizes to the “373” gene and has a chain length of at least 15 nucleotides is used.
  • the term “specifically hybridizes” as used herein refers to the ability to cross-hybridize with MA and / or RNA or other genes encoding other genes under ordinary hybridization conditions, preferably under stringent hybridization conditions. It means that the dimensioning does not occur significantly.
  • the probe and the transfer membrane are hybridized at 68 in Express Hydridat on Solution (manufactured by CL0NTECH), and finally 0.1 X SSC, 0.05% SDS Stringent conditions can be achieved by washing with a solution at 50.
  • nucleic acid molecules may be synthetic or natural.
  • Hybrida Usually, a labeled DNA is used as the probe DNA used in the experiment. Labels include, for example, nick translation labeling using DNA polymerase 1, end labeling using polynucleotide kinase, fill-in labeling using Klenow fragment (Berger SL, Kimmel AR. (1987) Guide to Molecular Cloning Techniques , Method in Enzymology, Academic Press; Hames BD, Higg ins SJ (1985) Genes Probes: A Practical Approach. IRL Press; Sambrook J, Fritsch EF, Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edn.
  • Testing of allergic diseases using the hybridization technique can be performed using, for example, a Northern hybridization method, a dot blot method, a method using a DNA microarray, and the like.
  • an RT-PCR method can be used as a method utilizing the gene amplification technique.
  • the expression of the “373” gene can be more accurately quantified by using a PCR amplification monitor method as shown in Example 8 in the process of gene amplification.
  • probes that are labeled with different fluorescent dyes at both ends to cancel each other's fluorescence are used to hybridize to the detection target (DNA or RNA reverse transcript).
  • the detection target DNA or RNA reverse transcript.
  • the two fluorescent dyes are separated and the fluorescence is detected. This fluorescence is detected in real time.
  • the number of copies of the target in the target sample can be determined at the same time as the number of cycles of PCR amplification by simultaneously measuring the standard sample whose copy number is clear for the target.
  • the test for an allergic disease of the present invention may be performed by detecting the protein encoded by “373”.
  • a Western blotting method using an antibody that binds to the protein encoded by “373”, an immunoprecipitation method, an ELISA method, and the like can be used.
  • Antibodies to the protein encoded by "373" of the present invention can be obtained as polyclonal antibodies or monoclonal antibodies using techniques well known to those skilled in the art (Milstein C, et al., 1983, Nature 305 (5934): 537-40).
  • the protein or its partial peptide to be used as an antigen is, for example, a 373 gene or a part thereof is incorporated into an expression vector, and this is introduced into an appropriate host cell to prepare a transformant. Is cultured to express a recombinant protein, and the expressed recombinant protein is purified from a culture or a culture supernatant.
  • the expression of the gene of the present invention if the expression of the gene of the present invention is significantly low, the subject can be determined to have a high allergen such as cedar pollen antigen and to have an allergic predisposition. .
  • the measurement of the expression level of the gene of the present invention in combination with the allergen-specific antibody titer, symptoms, and the like can be used for examination of allergic diseases.
  • the “373” gene expressed in T cells has high IgE specific to pollen antigens, and its expression is reduced in hay fever patients. Even in an allergic patient who exhibits responsiveness to an antigen other than cedar pollen, the expression of the “373” gene may be reduced while T cell responsiveness to the antigen is enhanced. In such cases, decreased expression of the “373” gene corresponds to increased T cell responsiveness, By monitoring the expression of the drug, screening for therapeutic drugs for allergic diseases can be performed.
  • the method for screening a candidate compound for treating an allergic disease 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 a transcript of “373” is obtained. Measure.
  • 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 “373” gene is measured.
  • the stimulation with the pollen antigen is performed for the purpose of inducing an antigen-specific allergic reaction in T cells and determining the therapeutic effect of the candidate compound on it.
  • peripheral blood lymphocytes are collected from hay fever humans or mice, and the peripheral blood lymphocytes are stimulated in vitro with cedar pollen antigen.
  • Candidate compounds are added during in vitro stimulation.
  • the T cells are then isolated from the stimulated peripheral blood lymphocytes and the transcript of “373” is measured. As a result of this measurement, a compound that increases the transcription amount of the “373” gene is selected.
  • Screening of the candidate compound for treating an allergic disease 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 a lymphocyte stimulator.
  • lymphocyte stimulants include calcium ionophore (A23187), PMA, phytohemagglutinin (PHA), and the like.
  • Add candidate drugs during in vitro stimulation Thereafter, the transcription amount of the “373” gene in the established T cells is measured. As a result of this measurement, a compound that increases the transcription of the “373” gene is selected.
  • Detection of the expression of the “373” gene in the screening of candidate compounds for the treatment of allergic diseases is carried out by the same method as the test for allergic diseases of the present invention.
  • the hybridization can be carried out using a hybridization technique using a nucleic acid to be probed, or a gene amplification technique using a DNA that hybridizes to the gene of the present invention as a primer.
  • a Northern hybridization method for example, a dot plot method, a method using a DNA microarray, or the like can be used.
  • a method utilizing the gene amplification technique an RT-PCR method can be used. In the RT-PCR method, more accurate quantification of the expression of the “373” 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 compound samples synthesized by existing chemical methods such as steroid derivatives, compound samples synthesized by combinatorial chemistry, extracts of animal and plant tissues, and microorganisms. A mixture containing a plurality of compounds such as a culture, a sample purified from them, and the like can be mentioned.
  • the compound isolated by the method for screening a candidate compound for a therapeutic drug for an allergic disease of the present invention is a candidate for a drug which improves allergic predisposition to an allergen such as a pollen antigen.
  • 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.
  • the screening of the present invention may be carried out by detecting the expression of the “373” protein in addition to detecting the expression of the “373” gene. That is, screening using the aforementioned “373” gene as an index Instead of using the “373” gene in the method, the “373” protein may be used as an index. In this case, the detection of the expression of the “373” protein is generally performed using an antibody that binds to the “373” protein.
  • This screening includes, for example, (a) a step of preparing T cells from a subject, (b) a step of preparing a protein sample from the T cells, and (c) binding of “373” protein in the protein sample to the protein. (D) selecting a compound that increases the amount of the protein to be detected as compared to a control (in the case where the test compound is not administered). it can.
  • a step of administering a test compound to a model animal of hay fever and stimulating it with a pollen antigen (b) a step of preparing T cells from the model animal, (c) preparing a protein sample from the T cells (D) detecting "373" protein in the protein sample using an antibody that binds to the protein; (e) comparing the control (when no test compound is administered) with the protein A step of selecting a compound that increases the detection amount of the compound.
  • An antibody that binds to the “373” protein used in this screening can be prepared by a known method.
  • a polyclonal antibody against the protein of the present invention is obtained by extracting blood of a mammal sensitized with an antigen and separating serum from the blood by a known method.
  • a serum containing the polyclonal antibody may be used. If necessary, a fraction containing the polyclonal antibody may be further isolated from this serum and used.
  • an immune cell may be obtained from a mammal sensitized with the above antigen, subjected to cell fusion to prepare a hybridoma, and an antibody produced by the hybridoma may be prepared.
  • the antibody When detecting the “373” protein, the antibody may be appropriately labeled and used. Further, without labeling the antibody, a substance that specifically binds to the antibody, for example, protein A or protein G may be labeled and detected. Examples of specific detection methods include: For example, the ELISA method can be used. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a diagram showing the antibody titers of cedar pollen-specific IgE antibodies in a total of 18 blood samples from 10 subjects who collected blood.
  • the value of cedar pollen-specific IgE antibody in each blood sample of subjects A to ⁇ was expressed in AU / ml.
  • the pair before pollen scattering is shown on the left (white column), and the one after scattering is shown on the right (black column).
  • Subjects A and B collected only blood after pollen scattering.
  • FIG. 2 is a graph showing changes in expression of “373” in a high IgE group and a normal IgE group when grouped according to cedar pollen-specific IgE values. Error bars represent standard deviation.
  • FIG. 3 is a diagram showing changes in the expression of “373” in the pre-scattering group and the post-scattering group when grouping was performed before and after the cedar pollen scattering time. Error bars represent standard deviation.
  • FIG. 4 is a photograph showing the results of Northern hybridization of “373” using mRNAs prepared from various cancer cell lines. BEST MODE FOR CARRYING OUT THE INVENTION
  • 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. The presence of atopic predisposition was determined by whether the value of the CAP RAST test for cedar pollen-specific IgE was greater than 2. That is, eight subjects A to G and I were regarded as atopic predisposition group (hereinafter also referred to as “patient”), and two subjects, subjects H and, were regarded as healthy subjects (hereinafter also referred to as “normal group”). Of the eight subjects with an atopic predisposition, 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 Nopo, etc., and blood was collected in a 10 ml syringe containing a final concentration of 50 unit / ml heparin. At this time, two 22G needles were prepared for one blood sample. The injection needle was removed and transferred to a 50 ml centrifuge tube (made of polypropylene). After centrifugation at 1500 rpm for 5 minutes at room temperature, 1.1 ml was collected from the surface as close as possible, and centrifuged at 15000 rpm for 5 minutes and at 4 to collect 1 ml of the supernatant as plasma.
  • the lymphocyte fraction obtained in Example 2 was centrifuged at 1200 rpm at 4 for 5 minutes, and suspended in BSA / PBS at 10 8 per 100 1. The capacity became about 201. This was transferred to an Eppendorf tube (1.5 ml), and the CD3 microbead solution was added. After that, it was left at 4-10 for 30 minutes (it was not placed on ice at this time). This sample was treated with a magnetic cell saw Yuichi (MACS) (manufactured by Miltenyi Biotech Inc.) as follows.
  • MCS magnetic cell saw Yuichi
  • the MS + / RS + column was attached to 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 drained. Next, cells labeled with CD3 microbeads were applied to the column. The column was washed three times with 500 il (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 force column. This was used as the T cell fraction.
  • the obtained T cell fraction was centrifuged at 1200 rpm for 5 minutes at 4t.
  • the precipitate was washed once with BSA / PBS. After the second wash, resuspend the cells in 1 ml and aliquot The number of cells was measured by diluting with blue two-fold. The total cell number was about 4 ⁇ 10 6 .
  • RNA from T cells was prepared using RNeasy Mini (manufactured by Qiagen) according to the attached manual in principle. All operations were performed at room temperature, wearing gloves. Four volumes of ethanol were added to Posh Buffer RPE. The lysis buffer RLT was supplemented with 10 l / ml 2-mercaptoethanol. The cell suspension was centrifuged at 1000-1200 rpm for 5 minutes, and the supernatant was removed by aspiration. To the precipitate was added 350 1 lysis buffer RLT (containing 2-mercaptoethanol) solution. At this stage, lysates of cells in RLT buffer could be stored at -70.
  • RNeasy Mini manufactured by Qiagen
  • the cell lysate was stored frozen, incubate at 37 for 10-15 minutes, and centrifuge for 3 minutes at maximum speed if insolubles were visible, collecting only the supernatant.
  • the lysate was homogenized with a syringe equipped with a 20 G force terran needle and then treated with Q IAshredder. (That is, usually, a lysate of 3501 cells was applied to a Kyaschlets unit using a Pitman. This was centrifuged at 1500 rpm for 2 minutes, and the effluent was collected.) 70% of 3501 Ethanol was added and mixed well by pipetting.
  • the RNeasy spin column was attached to the attached 2 ml tube, the lysate mixture of cells was applied, centrifuged at 8000 Xg (11500 rpm) for 1 minute, and the effluent was discarded.
  • Posh buffer RW1 700 1 was applied to the column, and it was put upright for 5 minutes. The mixture was centrifuged at 11,500 rpm for 15 seconds, and the effluent was discarded.
  • the column was placed in a new 2 ml tube, and Posh buffer RPE (containing ethanol) 500 1 was applied to the column. The mixture was centrifuged at 11,500 rpm for 15 seconds, and the effluent was discarded. Wash buffer RPE 500 1 was applied to the column and centrifuged at maximum speed for 2 minutes.
  • the column was placed in a new 1.5 ml tube, 30 ⁇ l of DEPC-treated water was applied, and the lid was capped and 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, re-attach the column to a new 1.5 ml tube, apply D EPC-treated water 30 1, cap the lid, stand for 10 minutes, and set the column at 11500 i "pm. Centrifuge for minutes.
  • Fluorescent differential display F1 uorescent Differential Display, abbreviated as “DD”) using total RNA prepared from T cells is described in the literature (T. Ito et al., 1994, FEBS Lett. 351: 231-236). Performed according to the method. Total RNA prepared from T cells was reverse transcribed to obtain cDNA.
  • cDNA was prepared using 0.2 g of total RNA for each of the three anchor primers.
  • cDNA was prepared using 0.4 // g RNA for each of the three anchor primers. All cDNAs were diluted to a final concentration of 0.4 ng / 1 RNA and used in the experiments.
  • a DD-PCR reaction was performed using cDNA equivalent to lng RNA per reaction. Table 1 shows the composition of the reaction solution. cDNA (0.4 ng / 1 RNA equivalent) 2.5 1
  • the PCR reaction conditions were as follows: 1 cycle of ⁇ 95 3 minutes, 5 minutes at 40, 5 minutes at 72 '', followed by 30 cycles of ⁇ 94 ⁇ 5 seconds, 2 minutes at 40, 1 minute at 72 '', followed by 72 cycles For 5 minutes and then continuously to 4.
  • the primer pairs used were the primer primers GT15A (SEQ ID NO: 3), GT 15C (SEQ ID NO: 4), and GT15G (SEQ ID NO: 5). 110110, AG 111-199, and AG 200-287 were combined, for a total of 287 sets of 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, and samples were applied and electrophoresed at 40 W for 210 minutes. Thereafter, the gel plate was scanned using Hitachi Fluorescence Image Analyzer -FMBI0 II, and electrophoretic images were obtained by fluorescence detection.
  • Example 7 Amplification of band excised by DD analysis and sequencing 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.
  • the band of “373” was found by DD analysis using GT15A (SEQ ID NO: 3) as an anchor primer and AG18 (AAGCTCTCGAZ SEQ ID NO: 6) as an arbitrary primer.
  • the gel containing the “373” band was cut out, stored in a TE solution, and heated at 60 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 190 bp was amplified.
  • GT15A was used as the primer and AG18 was used as the optional primer.
  • the amplified DNA fragment was cloned using a plasmid vector pCR2.1 (Invitrogen) to obtain a plasmid P373-18 having a DNA fragment of about 190 bp.
  • the nucleotide sequence of the DNA fragment was determined according to a conventional method.
  • the expression amount of “373” was quantified by the TaqMan method using ABI-PRI SM7700. This method uses a fluorescent dye to quantitatively detect the PCR-amplified DNA strand in real time.
  • RNA samples before and after cedar pollen scattering were collected from 22 volunteers in the spring of 1998, T cells were prepared, and total RNA was extracted. The expression level of the target gene was quantified using a total of 44 RNA samples.
  • primers P373f G GAMGATCGTCAGGAMCTGGZ SEQ ID NO: 7
  • P373r TCCCTTCMCAAGTCTGCCCZ SEQ ID NO: 8
  • TaqMan probe P373 CAGCATCATCATCAMCATGGCTTCCTTG NO SEQ ID NO: 9
  • the TaqMan probe P373 was fluorescently labeled at the 5 'end with FAM (6-carboxyfluorescein) and at the 3' end with TA RA (6-carboxy-tetramethyl-rhodamine).
  • reverse transcribed cDNA was used as a primer with poly T (12-18 mer) from 44 total RNAs.
  • a serial dilution of the plasmid P373-18 obtained in Example 7 was used to carry out the reaction.
  • Table 3 shows the composition of the reaction mixture for monitoring PCR amplification.
  • the same quantitative analysis was performed on the 3) -actin (/ 3-actin) gene, and correction was performed based on the copy number of those genes. The number of copies of the gene (373) was calculated.
  • Table 4 shows the number (copy number) of “373” in each sample corrected for the copy number of 3-actin. For the correction, the average copy of 3-actin) in all samples was obtained, and the copy number of “373” in each sample was divided by the relative value of 3-actin in each sample when that was set to 1. Table 4
  • the grouping was performed before and after scattering of Japanese cedar pollen, or at least 3.5 AU / ml for each specific serum IgE in serum (high IgE group) and other (normal). (IgE group). For example, in the case of cedar pollen, the number of individuals in each group was 10 in the high IgE group and 12 in the normal IgE group. The test was performed separately for the group showing 200 AU / ml for total IgE and the other groups. Two-way analysis of variance was tested using StatView software (Abacuus Concepts, Inc.).
  • CL0NTECH Human Immune System MTN Blot II and Human Cancer Cell Line MTN Blot both membranes already transcribed mRNA were used.
  • the probe DNA was labeled with 32 P using a Random Primer Labeling Kit (TAKARA).
  • Northern hybridization and membrane washing were performed using Express Hybridization Solution (CLONTECH) according to the instructions attached.
  • Washing was performed three times at room temperature using 2xSSC-0.13 ⁇ 4SDS while measuring the radioactivity of the membrane with a Geiger counter. When the count reached 1000 or more and tens of thousands or less under the condition of being in close contact with the membrane, it was exposed to an imaging plate to acquire an image. Images were acquired with a Molecular Imager System (BIO-RAD). As a result of Northern analysis of “373”, a main band of 7.5 kb and a sub-band of 6.7 kb were detected, and it was inferred that “373” of the present invention was expressed as an approximately 7.5 kb mRNA (FIG. 4). ).
  • a database search of the nucleotide sequence of "373” revealed that there were multiple ESTs with homology. Therefore, after extracting an EST sequence having homology to “373” from dbEST, the individual sequences were assembled using ABI AutoAssembler to obtain a 5.6 kb sequence.
  • An open reading frame (0RF) consisting of 1324 amino acids was predicted in this sequence, but the absence of the 5 'non-coding region and the result of Northern hybridization revealed that the predicted mRNA size was 7.5%. Since it was kb, 5'-RACE was performed next, and the further upstream sequence was determined.
  • 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 for whether or not the subject has an allergic predisposition and a screening for candidate compounds for a therapeutic drug for allergic diseases.

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Abstract

L'invention concerne un nouveau gène, caractérisé par un niveau d'expression particulièrement faible chez les sujets présentant des taux élevés d'IgE spécifique au pollen de cèdre, et que l'on a réussi à isoler par la préparation de lymphocytes T à partir de sujets présentant différents taux d'IgE spécifique au pollen de cèdre avant et après la saison des pollens, et par la recherche dudit gène selon la technique de l'affichage différentiel. On a découvert que ce gène pouvait être utilisé dans l'examen des allergies et le criblage de composés candidats pour des remèdes contre les allergies.
PCT/JP2000/002730 1999-04-27 2000-04-26 Gene 373 associe a la pollinose Ceased WO2000065046A1 (fr)

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JP12048999A JP2003125775A (ja) 1999-04-27 1999-04-27 花粉症関連遺伝子、３７３

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024903A1 (fr) * 2000-09-25 2002-03-28 Genox Research, Inc. Methode d'examen applicable en cas de maladie allergique
WO2002050269A1 (fr) * 2000-12-21 2002-06-27 Genox Research, Inc. Methode d'etude de maladies allergiques
WO2017146011A1 (fr) * 2016-02-22 2017-08-31 国立大学法人 千葉大学 Biomarqueur pour le diagnostic de la rhinite allergique

Citations (1)

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Publication number Priority date Publication date Assignee Title
JPH11332567A (ja) * 1998-05-22 1999-12-07 Dai Ichi Seiyaku Co Ltd アトピー体質の判定方法

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JPH11332567A (ja) * 1998-05-22 1999-12-07 Dai Ichi Seiyaku Co Ltd アトピー体質の判定方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO2002050269A1 (fr) * 2000-12-21 2002-06-27 Genox Research, Inc. Methode d'etude de maladies allergiques
WO2017146011A1 (fr) * 2016-02-22 2017-08-31 国立大学法人 千葉大学 Biomarqueur pour le diagnostic de la rhinite allergique
JPWO2017146011A1 (ja) * 2016-02-22 2019-01-17 国立大学法人千葉大学 アレルギー性鼻炎の診断用バイオマーカー

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