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WO2000065048A1 - Gene 581 associe a la pollinose - Google Patents

Gene 581 associe a la pollinose Download PDF

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Publication number
WO2000065048A1
WO2000065048A1 PCT/JP2000/002732 JP0002732W WO0065048A1 WO 2000065048 A1 WO2000065048 A1 WO 2000065048A1 JP 0002732 W JP0002732 W JP 0002732W WO 0065048 A1 WO0065048 A1 WO 0065048A1
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Prior art keywords
gene
expression
seq
cells
allergic
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English (en)
Japanese (ja)
Inventor
Takeshi Nagasu
Yuji Sugita
Tomoko Kashiwabara
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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Publication of WO2000065048A1 publication Critical patent/WO2000065048A1/fr
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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
    • 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

  • 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 present inventors have proposed a method for preparing a plurality of humans based on the already established procedure of the “fluorescent DD (Fluorescent DD) method” (T. I to et al., 1994, FEBS Lett.
  • 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 “581” 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 exhibiting 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, the present invention relates to the following nucleic acid molecules, a method for testing an allergic disease based on the nucleic acid molecule, and a method for screening a candidate compound for a therapeutic drug for an allergic disease.
  • nucleic acid molecule comprising the protein coding region of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 11;
  • nucleic acid molecule according to any one of the following (a) to (c):
  • a protein comprising the amino acid sequence of SEQ ID NO: 12, in which one or several amino acids consist of a substituted, deleted, inserted, and / or added amino acid sequence;
  • [3] A protein encoded by the nucleic acid molecule according to [1] or [2].
  • [4] A vector into which the nucleic acid molecule according to [1] or [2] has been inserted.
  • [5] A transformant that retains the nucleic acid molecule of [1] or [2] or the vector of [4] in an expressible manner.
  • [6] The method for producing the protein according to [3], comprising culturing the transformant according to [5] and collecting an expression product.
  • a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 11, or a polynucleotide that hybridizes with a complementary strand thereof, and has a chain length of at least 15 nucleotides .
  • [12] allergy including the step of measuring the expression level of a gene consisting of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 11 in a biological sample and comparing it with a control (in the case of a healthy subject) How to test for disease.
  • the biological sample is blood, and the expression level of the gene is determined by measuring a protein comprising the amino acid sequence shown in SEQ ID NO: 12 and / or a fragment thereof in the blood. [12] the method of.
  • a method for screening a candidate compound for a therapeutic drug for an allergic disease comprising:
  • step (c) selecting a compound that increases the expression level of the gene measured in step (b) as compared to a control (in the case where no test compound is administered).
  • step (f) selecting a compound that increases the expression level of the gene measured in step (e) as compared to a control (in the case where no test compound is administered).
  • step (e) In step (d), compared to the control (when no test compound is administered) Selecting a compound that increases the expression level of the gene to be measured.
  • step (c) selecting a compound that increases the expression level of the gene measured in step (b) as compared to a control (in the case where no test compound is administered).
  • lymphocytes are prepared from peripheral blood
  • 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 allergic diseases can include bronchial asthma, allergic rhinitis, atopic dermatitis, hay fever, or insect allergy.
  • Allergic diathesi s is a genetic factor transmitted from a parent with an allergic disease to a child. Allergic diseases that occur familially are also called atopic diseases, and the genetic factors that cause them are atopic predispositions.
  • 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 expression level of the gene in the present invention is used as a term including either the amount of transcribed mRNA or the amount of a protein that is a translation product based on this mRNA.
  • the present invention relates to a novel gene “58 1” correlated with an IgE production response to cedar pollen of an individual.
  • the nucleotide sequence of the “581” cDNA found by the present inventors is shown in SEQ ID NO: 1.
  • the nucleotide sequence of the “581” cDNA isolated by the present inventors is a partial distribution sequence of the “581” cDNA, and those skilled in the art may use the sequence information of the “581” cDNA described in SEQ ID NO: 1. Alternatively, isolation of the full-length cDNA of "581" can be routinely performed. That is, a method for screening a T cell cDNA library or the like by hybridization using a sequence derived from “581” as a probe, or a method for screening DNA such as a T cell cDNA library using a sequence derived from “581” as a primer.
  • the full-length nucleotide sequence of “581” according to the present invention can be determined based on many ESTs and genomic information to be determined in the future.
  • the nucleotide sequence shown in SEQ ID NO: 1 has the same sequence as the 5 'end of KIAA1097 consisting of about 4.3 kb as a result of homology search. I understood. That is, the nucleotide sequence shown in SEQ ID NO: 1 was included in KIAA1097. Although no initiation codon is found in KIAA1097, an ORF of 981 amino acids is estimated.
  • KIAA1097 is an EST published after the present inventors determined the nucleotide sequence shown in SEQ ID NO: 1.
  • nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 11” according to the present invention includes a single nucleotide based on the sequence information of “581” cDNA described in SEQ ID NO: 1. Includes the releasable "581" full-length cDNA.
  • the nucleic acid molecule of the present invention can hybridize not only to the nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 11 but also to these nucleic acid molecules under stringent conditions. Includes nucleic acid molecules that encode proteins functionally equivalent to number 12.
  • the stringent conditions include, for example, the following conditions. Hybridization conditions can be adjusted according to the length of the nucleic acid molecule and the constituent bases.
  • the functionally equivalent to “581” means that the atopic predisposing group (1 ⁇ value for cedar pollen is 3.5 l / ml or more) is significantly more significant than the non-atopic predisposing group. Indicates low expression. Nucleic acid molecules that can hybridize under stringent conditions are likely to encode proteins with high structural homology. Thus, a nucleic acid molecule encoding a protein functionally equivalent to “58 1” in can be obtained.
  • nucleic acid molecule of the present invention comprises an amino acid sequence shown in SEQ ID NO: 12 in which one or several amino acids have been substituted, deleted, inserted, and / or added. And a nucleic acid molecule encoding a protein functionally equivalent to the protein consisting of the amino acid sequence described in (1).
  • the nucleic acid molecule of the present invention can be obtained by a known method based on SEQ ID NO: 1 or the base sequence information described in SEQ ID NO: 11.
  • the desired cDNA can be selected by screening a human T cell cDNA library with a probe set based on the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 11.
  • the target gene can also be amplified by performing PCR using primers set based on SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 11. If cDNA derived from lymphocytes of non-human animals is used as a library for screening or a template for PCR, homologs of “581” in other species can be obtained.
  • nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 11 can also be chemically synthesized.
  • Known methods (Sambrook II, Fritsch, EF, and Mania Us T. Molecular cloning: A Laboratory Manual (2nd edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor.) Can be used.
  • the nucleic acid molecule of the present invention thus obtained is useful for producing the protein “58 1” of the present invention as a recombinant.
  • the protein of the present invention obtained as a recombinant can be used as an immunogen for obtaining an antibody against “581”.
  • the present invention relates to a polynucleotide that hybridizes with a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 11, or a complementary strand thereof, and has a chain length of at least 15 nucleotides.
  • a polynucleotide that can hybridize to a given base sequence.
  • probes and primers that specifically hybridize to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 11 as well as hybridizable to an unknown nucleotide sequence that is structurally similar are designed. Methods are also known.
  • a polynucleotide having the designed base sequence can be chemically synthesized by a technique such as the dideoxy method.
  • a polynucleotide comprising the required nucleotide sequence can be obtained by cleaving the cDNA clone with an appropriate restriction enzyme. These polynucleotides are useful as probes and primers for detecting and synthesizing the nucleic acid molecule of the present invention.
  • “581” showed significantly lower expression in the atopic predisposing group (IgE value for cedar pollen was 3.5 AU / ml or more) than in the non-atopic predisposing group. Therefore, it is possible to test for allergic diseases and screen candidate compounds for the treatment of allergic diseases using the expression of the “581” gene (including transcription into mRNA and translation into protein) as an index. it is conceivable that.
  • cedar pollinosis is particularly preferred as an allergic disease to be tested and treated.
  • Detection of the expression of the “581” gene in the test for allergic disease can be performed by a hybridization technique using a nucleic acid that hybridizes to the “581” gene as a probe, or a primer using a DNA that hybridizes to the gene of the present invention. It is possible to use the gene amplification technology described above.
  • a nucleic acid molecule that specifically hybridizes to the “581” gene and has a chain length of at least 15 nucleotides is used.
  • “specifically hybridize” means a normal hybrid. It refers to the fact that under hybridization conditions, preferably under stringent hybridization conditions, there is no significant cross-hybridization with DNA and / or RNA encoding other genes.
  • the probe and the transfer membrane are hybridized in Express Hybridization Solution (manufactured by CL0NTECH) at 68: and finally washed with 50 in 0.1X SSC, 0.05% SDS solution. Stringent conditions can be used.
  • nucleic acid molecules used in the test of the present invention may be synthetic or natural.
  • the probe DNA used for hybridization is usually labeled.
  • Labels include, for example, nick translation labeling using DNA polymerase 1, terminal labeling using polynucleotide kinase, and fill-in terminal labeling using Klenow fragment (Berger SL, Ki band el AR. (1987) Guide to Molecular Cloning Techniques, Method in Enzymology, Academic Press; Hames BD, Higgins SJ (1985) Genes Probes: A Practical Approach.IR L Press; Sambrook J, Fritsch EF, Maniat is T. (1989) Molecular Cloning: a Laboratory Manual , 2nd Edn.
  • an RT-PCR method can be used as a method using gene amplification technology.
  • the expression of the “581” 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 at both ends to cancel each other's fluorescence are used to hybridize to the detection target (reverse transcript of DNA or RNA).
  • the detection target reverse transcript of DNA or RNA
  • the number of copies of the target to be detected 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. USA 88:72 76-7280; Livak, KJ et al., 1995, PCR Methods and Applications 4 (6): 35 7-362; Heid, CA et al., Genome Research 6: 986. -994; Gibson, EMU et al., 1996, Genome Research 6: 995-1001).
  • ABI PRISM7700 PerkinElmer
  • the test for an allergic disease of the present invention may be performed by detecting the protein encoded by “581”.
  • a test method for example, a Western blotting method using an antibody that binds to a protein encoded by “581”, an immunoprecipitation method, an ELISA method, and the like can be used.
  • a sample for detecting protein a lysate of T cells or a blood sample such as serum or plasma can be used.
  • a whole blood sample containing T cells can be lysed to prepare a sample.
  • Antibodies to the protein encoded by "581" of the present invention can be obtained as polyclonal 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 used as an immunogen in the present invention may be, for example, a peptide consisting of an amino acid sequence selected from the amino acid sequences described in SEQ ID NO: 12.
  • the protein or its partial peptide used for the antigen is prepared by incorporating the “581” gene or a part thereof into an expression vector, introducing this into an appropriate host cell, and performing transformation. It can be obtained by preparing a recombinant, culturing the transformant to express a recombinant protein, and purifying the expressed recombinant protein from a culture or a culture supernatant.
  • 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 “581” gene expressed in T cells has high IgE specific for pollen antigen, and its expression is reduced in a group of hay fever patients. Even in an allergic patient who exhibits responsiveness to an antigen other than cedar pollen, the expression of the “581” gene may be reduced while T cell responsiveness to the antigen is enhanced. In such cases, a decrease in the expression of the “581” gene corresponds to an increase in T cell responsiveness, and therefore, monitoring of the expression of the “581” gene enables screening for therapeutic drugs for allergic diseases. .
  • 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 the transcript of “581” 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 “581” gene is measured.
  • a compound that increases the transcription amount of the “581” gene is selected.
  • 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 a mouse or the like, 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 “581” transcript is measured.
  • a compound that increases the transcription amount of the “581” 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 stimulating substances include calcium ionophore (A23187), PMA, and phytohemagglutinin (PHA).
  • Candidate drugs are added during in vitro stimulation. Thereafter, the transcription amount of the “581” gene in the established T cells is measured. As a result of this measurement, a compound that increases the transcription of the “581” gene is selected.
  • Detection of the expression of the “581” gene in the screening of candidate compounds for the treatment of allergic diseases is performed by the hybridization technique using a nucleic acid that hybridizes to the “581” gene as a probe, as in the test for allergic diseases of the present invention.
  • it can be carried out using a gene amplification technique using DNA that hybridizes to the gene of the present invention as a primer.
  • a Northern hybridization method for example, 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 the gene amplification technique.
  • the expression of the “581” gene can be more accurately quantified by using a PCR amplification monitor method as shown in Example 8 in the gene amplification process.
  • Operations such as separation of lymphocytes and T cells, extraction of RNA from T cells, and synthesis of cDNA can be performed according to known techniques as described in Examples.
  • test compounds used in these screenings include compound preparations synthesized by existing chemical methods such as steroid derivatives, and combinatorial chemistry.
  • a mixture containing a plurality of compounds such as an animal or plant tissue extract or a microbial culture, and a preparation purified therefrom may 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.
  • 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 ⁇ to ⁇ was expressed in ⁇ / 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 diagram showing changes in the expression of “581” in a high IgE group and a normal IgE group when classified according to cedar pollen-specific IgE values. Error bars represent standard deviation.
  • FIG. 3 is a graph showing changes in the expression of “581” 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. 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, subjects A to G and I were eight subjects as atopic predisposition group (hereinafter also referred to as “patient”), and 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 treated with 1 ml of Heparin from Nopo, etc. without any restriction, 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).
  • PRP PRP (Pla teletrich plasma, platelet-rich plasma) was transferred to another 15 ml centrifuge tube, and centrifuged at 1200 rpm (equivalent to 150 Xg in a Tommy centrifuge) for 5 minutes at room temperature. After centrifugation, platelets were in the supernatant. 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 pipe.
  • the mixture was centrifuged at 1500 rpm (equivalent to 400 Xg in a Tomy centrifuge) for 30 minutes at room temperature.
  • granulocytes and erythrocytes precipitated, and lymphocytes, monocytes, and platelets were contained in the middle layer with the ficoll layer in between.
  • the lymphocyte fraction obtained in Example 2 was centrifuged at 1200 ⁇ ⁇ ⁇ at 4 for 5 minutes, and suspended in BSA / PBS to a concentration of about 100 1. The capacity became about. 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 sorter (MACS) (Miltenyi Biotech In) as follows. 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 poured out.
  • MCS magnetic cell sorter
  • the obtained T cell fraction was centrifuged at 1,200 rpm at 4 for 5 minutes.
  • the precipitate was washed twice with BSA / PBS. After the first washing, the cells were suspended in 1 ml, and a part thereof was diluted 2-fold with trypan blue, and the cell number was measured. Total cell number was approximately 4 ⁇ 10 6 .
  • RNA from T cells was prepared using RNeasy Mini (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. Lysis buffer RLT was supplemented with 10 1 / ml 2-mercaptoethanol. The cell suspension was centrifuged at 1000-1200 ⁇ ⁇ ⁇ for 5 minutes, and the supernatant was removed with an aspirator. A lysis buffer RLT (containing 2-mercaptoethanol) solution was added to the precipitate. At this stage, lysates of cells in RLT buffer could be stored at -70.
  • RNeasy Mini 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 20G force teran needle and then treated with Q IAshredder. (That is, usually, 350/1 cell lysate was applied to a Kyaschlets dunit using a Pitman. This was centrifuged at 1500 ⁇ 01 for 2 minutes, and the effluent was collected.) % Ethanol was added and mixed well by pipetting.
  • DNase treatment was performed to remove DNA from total RNA prepared from T cells.
  • DD Fluorescent differential display using total RNA prepared from T cells
  • the PCR reaction conditions were 95 cycles of 3 minutes, 40 cycles of 5 minutes, and 72 cycles of 5 minutes for one cycle, followed by 30 cycles of 9415 seconds, 40 cycles of 2 minutes, and 72 1 minutes. It was 5 minutes at 72 and then continuously at 4.
  • the primer pairs used are the anchor primers GT15A (SEQ ID NO: 2), GT A total of 287 reactions were carried out using 15C (SEQ ID NO: 3) and GT15G (SEQ ID NO: 4) with optional primers in combination with AG1-110, AG111-199, and AG200-287, respectively.
  • an optional primer 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 I I, and electrophoresis images were obtained by fluorescence detection.
  • 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 “581” was found by DD analysis using GT15C (SEQ ID NO: 3) as an anchor primer and AG164 (CATTCTCAGG / SEQ ID NO: 5) as an arbitrary primer, and the expression of “581” was found in Japanese cedar. A difference was observed between the group with high specific IgE (atopic predisposition group) and the normal group.
  • a gel containing the band of “581” was cut out, stored in a TE solution, and heated at 60:10 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 260 bp was amplified.
  • GT15C was used as the primer and AG164 was used as the optional primer.
  • the amplified DNA fragment was cloned with a plasmid vector pCR2.1 (Invitrogen) to obtain a plasmid p58-23 carrying a DNA fragment of about 260 bp.
  • a plasmid DNA the nucleotide sequence of the DNA fragment was determined according to a conventional method.
  • the expression amount of “581” was quantified by the TaqMan method using ABI-PRISM7700. This method quantitatively detects the PCR-amplified DNA strand in real time using a fluorescent dye. System.
  • 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.
  • V fly ( ⁇ 0.34 ⁇ 0.34 0.79 0.81 180 fly tt 0.34 ⁇ 0.34 0.78 0.9 160
  • primers 5 ' (TGAGTTCCTAATTTCCTATCCAAAAATAC SEQ ID NO: 6), 581-3' (GCATTATCTAACTGTG AACCTTACCACTZ SEQ ID NO: 7), and TaqMan probe 581SEQS5 (TCTTTGCTGCAA GCATAACACCATACTCGA / SEQ ID NO: 8) was designed, synthesized, and used for the quantitative reaction.
  • TaqMan probe 581SEQS5 was fluorescently labeled at the 5 'end with FAM (6-carboxyfluorescein) and at the 3' end with TAMRA (6-carboxy-tetramethytriodamine).
  • FAM 6-carboxyfluorescein
  • TAMRA 6-carboxy-tetramethytriodamine
  • type I cDNA obtained by reverse transcription of poly T (12-18 mer) as a primer from all 44 RNAs was used.
  • a serial dilution of the plasmid p58-23 obtained in Example 7 was used to carry out the reaction.
  • Table 3 shows the composition of the reaction solution for monitoring PCR amplification.
  • the same quantitative analysis was performed for the ⁇ -actin (j3-actin) gene, and the correction was performed based on the copy number of those genes. ) was calculated.
  • Table 4 shows the number (copy number) of “581” in each sample corrected for the copy number of) 3-actin. For the correction, the average copy of i3-actin in all samples was determined, and the number of copies of “581” in each sample was divided by the relative value of 3-actin (in each sample assuming that it was 1).
  • V Flying M Before 286 Flying K After 121 Using this value, two-way analysis of variance was performed.
  • the groups were divided into groups that showed 3.5 AU / ml or more before and after cedar pollen scattering or at least one of the two measurements of serum specific IgE (high IgE group) and other groups (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.).
  • 5 'RACE System for Rapid Amplification of cDNA Ends V.2 kit (BRL) was used. Furthermore, amplification was performed by PCR using a primer containing a 581-RT1 primer and a poly G adapter, to prepare double-stranded DNA. Using this as a type I, PCR was performed with 58 u1 (CTGAGTTCCAAATTTCCTATCC SEQ ID NO: 10), which is a primer specific to "581," and the AUAP primer in the adapter. The amplified DNA was subjected to sub-cloning and the nucleotide sequence was determined. As a result, a sequence of about 0.7 kb including the DD sequence was obtained. C This sequence is shown in SEQ ID NO: 1. Industrial applicability
  • 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 an examination as to whether or not it has an allergic predisposition and to screen a candidate drug for a therapeutic agent for an allergic disease.
  • AAAAADAA CC CCDBBBBBB CCCCCCCBB
  • MMGU A UYKT AGYHN EER GL2BFRFZE JI T cells were prepared from multiple subjects with different cedar pollen-specific IgE values before and after pollen scattering, and genes were searched by differential display. We succeeded in isolating a novel gene with significantly lower expression in subjects. The present inventors have found that this gene can be used for testing allergic diseases and for screening candidate compounds for therapeutic drugs for allergic diseases.
  • a nucleic acid molecule comprising the protein coding region of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 11.
  • a nucleic acid molecule according to any one of the following (a) to (c):
  • a protein comprising the amino acid sequence of SEQ ID NO: 12, comprising one or more amino acid substitution, deletion, insertion, and / or addition of an amino acid sequence;
  • a nucleic acid molecule encoding a protein functionally equivalent to the protein consisting of the described amino acid sequence;
  • a vector into which the nucleic acid molecule according to claim 1 or 2 has been inserted is inserted.
  • a method for producing the protein according to claim 3, comprising a step of culturing the transformant according to claim 5 and collecting an expression product.
  • a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 11, or a polynucleotide that hybridizes with a complementary strand thereof, having a chain length of at least 15 nucleotides.
  • Claim 1 or Claim 2 consisting of the polynucleotide of Claim 7.
  • a method for testing an allergic disease comprising the step of measuring the expression level of a gene consisting of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 11 in a biological sample and comparing it with a control (in the case of a healthy subject) .
  • the biological sample is a T cell
  • the expression level of the gene in the T cell is measured by RT-PCR using mRNA as a type III.
  • a method for screening a candidate compound for a therapeutic agent for an allergic disease comprising:
  • step (c) selecting a compound that increases the expression level of the gene measured in step (b) compared to a control (in the case where the test compound is not administered).
  • a method for screening a candidate compound for a therapeutic agent for an allergic disease comprising:
  • step (f) selecting a compound that increases the expression level of the gene 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 an allergic disease comprising:
  • step (e) selecting a compound that increases the expression level of the gene measured in step (d) 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 an allergic disease comprising:
  • step (b) measuring the expression level of the gene comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 11 in the established T cells, (c) selecting a compound that increases the expression level of the gene measured in step (b) as compared to a control (in the case where no test compound is administered).
  • T cells are prepared from peripheral blood of a model animal of hay fever.
  • lymphocytes are prepared from peripheral blood.

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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/JP2000/002732 1999-04-27 2000-04-26 Gene 581 associe a la pollinose Ceased WO2000065048A1 (fr)

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JP12049299A JP2003125777A (ja) 1999-04-27 1999-04-27 花粉症関連遺伝子、５８１

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Citations (2)

* Cited by examiner, † Cited by third party
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
WO2000009709A2 (fr) * 1998-08-10 2000-02-24 Incyte Pharmaceuticals, Inc. Proteases et proteines associees

Patent Citations (2)

* Cited by examiner, † Cited by third party
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
WO2000009709A2 (fr) * 1998-08-10 2000-02-24 Incyte Pharmaceuticals, Inc. Proteases et proteines associees

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK KIKUNO REIKO ET AL.: "Prediction of the coding sequences of unidentified human genes. XIV. The complete sequences of 100 new cDNA clones from brain which code for large protein in vitro" *
HOLROYD KENNETH J. ET AL.: "Asthma and bronchial hyperresponsiveness linked to the XY long arm pseudoautosomal region", GENOMICS, vol. 52, no. 2, 1 September 1998 (1998-09-01), pages 233 - 235, XP002930133 *
KAZUSHI HONDA: "Sugi kafun sho no meneki idengaku teki kaiseki HLA to rensa shita sugi kafun kogen ni taisuru meneki yokusei idenshi no shoumei to kaiseki", FUKUOKA IGAKU ZASSHI, vol. 80, no. 1, 25 January 1989 (1989-01-25), pages 28 - 37, XP002946664 *
KIKUNO, DNA RESEARCH, vol. 6, no. 3, 30 June 1999 (1999-06-30), pages 197 - 205, XP002930136 *
MITSURU MUNAKATA: "beta-Adrenalin juyotai idenshi", GENDAI IRYO, vol. 30, no. 3, 10 March 1998 (1998-03-10), pages 863 - 867, XP002946665 *
TAKABAYASHI AKIRA ET AL.: "Novel polymorphism in the 5'-untranslated region of the interleukin-4 gene", JOURNAL OF HUMAN GENETICS, vol. 44, no. 5, 1999, pages 352 - 353, XP002930134 *

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