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WO2000073440A1 - Gene 787 associe a la pollinose - Google Patents

Gene 787 associe a la pollinose Download PDF

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Publication number
WO2000073440A1
WO2000073440A1 PCT/JP2000/003192 JP0003192W WO0073440A1 WO 2000073440 A1 WO2000073440 A1 WO 2000073440A1 JP 0003192 W JP0003192 W JP 0003192W WO 0073440 A1 WO0073440 A1 WO 0073440A1
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Prior art keywords
cells
preparing
compound
antigen
suppresses
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English (en)
Japanese (ja)
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WO2000073440A8 (fr
Inventor
Takeshi Nagasu
Yuji Sugita
Tomoko Fujishima
Tadahiro Oshida
Masaya Obayashi
Shigemichi Gunji
Izumi Obayashi
Yukiho Imai
Nei Yoshida
Kaoru Ogawa
Keiko Matsui
Eiki Takahashi
Akira Yokoi
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Eisai Co Ltd
Genox Research Inc
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Eisai Co Ltd
Genox Research Inc
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Publication of WO2000073440A8 publication Critical patent/WO2000073440A8/fr
Anticipated expiration legal-status Critical
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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
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • 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 antigen stimulatory response, a method for testing an allergic disease using the expression of the gene as an index, and a method for screening a candidate therapeutic compound for suppressing a T cell antigen stimulatory response.
  • 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.
  • 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.
  • Allergic diseases such as hay fever are one of the diseases seen by many people in recent years.
  • the pathogenesis of hay fever may be related to several genes whose expression is affected by pollen, one of the environmental factors. Under such circumstances, it has been desired to isolate genes associated with allergic diseases such as hay fever. Disclosure of the invention
  • An object of the present invention is to provide a gene associated with an allergic disease. Further, the present invention provides a method for testing an allergic disease, using the expression of the gene as an index.
  • the present inventors have proposed a method for treating a plurality of humans based on the already established “Fluorescent DD (Fluorescent DD) method” (T. I. to 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 in a plurality of subjects including pollinosis patients and expressed them between subjects with different cedar pollen-specific IgE values and before and after pollen dispersal.
  • the present inventors performed a comparative analysis of the expression level of the isolated 787J gene in lymphocytes isolated from subjects before and after pollen scattering, and found that the gene showed a significantly lower value after cedar pollen scattering. Therefore, the present inventors used the expression level of the gene as an indicator to test for allergic diseases and to suppress antigen-stimulated response of T cells. It has been found that screening of drug candidate compounds is possible.
  • the present invention relates to a gene showing a low value after pollen scattering, a method for testing allergic disease using the expression of the gene as an index, and a method for screening a candidate therapeutic compound for suppressing T cell antigen stimulation response. More specifically,
  • nucleic acid molecule comprising the base sequence of SEQ ID NO: 20,
  • nucleic acid molecule comprising the protein coding region of the nucleotide sequence of SEQ ID NO: 20, [3] a nucleic acid molecule specifically hybridized to the nucleic acid molecule of [1] or [2], and a chain length of at least 15 nucleotides DNA having
  • T cells are prepared from peripheral blood of the subject
  • step (f) selecting a compound that suppresses the decrease in the amount of RNA measured in step (e) as compared to a control (in the case where the test compound is not administered);
  • step (f) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (e) as compared to a control (in the case where no test compound is administered); [12] A method for screening a candidate therapeutic compound that suppresses the antigen-stimulated response of T cells,
  • step (h) selecting a compound that suppresses the decrease in the amount of RNA measured in step (g) compared to a control (in the case where the test compound is not administered),
  • step (h) selecting a compound that suppresses a decrease in 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 suppresses the decrease in the amount of RNA measured in step (: f) as compared to a control (in the case where no test compound is administered);
  • step (g) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (f) as compared to a control (in the case where the test compound is not administered);
  • step (e) selecting a compound that suppresses the decrease in the amount of RNA measured in step (d) compared to a control (in the case where the test compound is not administered),
  • step (e) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (d) 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 is the allele Means that T cells show an immune response upon the stimulation of the gene.
  • Typical allergic diseases can include bronchial asthma, allergic rhinitis, atopic dermatitis, hay fever, or insect allergy.
  • Allergic predisposition 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 predispositions.
  • nucleic acid molecule in the present invention includes DNA and RNA.
  • the present invention relates to a novel gene “787J” correlated with the response of lymphocytes to cedar pollen antigen.
  • the nucleotide sequence of the “787” cDNA found by the present inventors is shown in SEQ ID NO: 20.
  • the nucleotide sequence of the “787” cDNA isolated by the present inventors is a partial distribution sequence of the “787” cDNA, and those skilled in the art will recognize the sequence information of the “787” cDNA described in SEQ ID NO: 20. Isolation of full-length cDNA for “787” based on the information 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 “787” as a probe, or a method for screening a T cell cDNA library for a T cell cDNA library or the like using a sequence derived from “787” as a primer.
  • nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 20” in the present invention includes “78” which can be isolated based on the sequence information of “787” cDNA described in SEQ ID NO: 20 as described above. 7 "full-length cDNA.
  • “787” indicates that lymphocytes in subjects were more consistent after pollen antigen exposure than before exposure. The expression was statistically significantly lower. Therefore, using the expression of the “787” gene (including transcription into mRNA and translation into protein) as an index, testing for allergic diseases and screening for candidate therapeutic compounds that suppress T cell antigen stimulation response It is considered possible. Decreased expression of “787” indicates the response of T cells to stimulation of antigens such as pollen.In patients with a history of allergic disease, expression of “787” is used as an index to determine the response of the patient to specific antigen exposure. Monitoring the transition is helpful in understanding the state of the disease and examining response to treatment.
  • cedar pollinosis is particularly preferred.
  • Detection of the expression of the "787" gene in the test for an allergic disease can be performed by using an hybridization technique using a nucleic acid that hybridizes to the "787" gene as a probe, or by hybridizing to the gene of the present invention. It can be performed using gene amplification technology using DNA as a primer.
  • a nucleic acid molecule that specifically hybridizes to the “787” gene and has a chain length of at least 15 nucleotides is used.
  • the term “specifically hybridizes” as used herein means that under normal hybridization conditions, preferably under stringent hybridization conditions, DNA and / or Z or RNA encoding other genes are cross-hybridized. Indicates that it does not occur significantly.
  • the probe and the transfer membrane were hybridized at 68 in Express Hydidi zat ion on Solut ion (manufactured by CL0NTECH), and finally, 0.1 X SS 0.05% SDS solution was used. Stringent conditions can be achieved by washing at ° C.
  • the probe DNA used for hybridization is usually labeled.
  • Labels include, for example, nick translation labeling using DNA polymerase I, end labeling using polynucleotide kinase, Fill-in labeling with Reno 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. IRL . Press; Sambrook J, Fr i tsch EF, Maniat is T.
  • the RT-KR method can be used as a method using gene amplification technology.
  • the expression of the “787” gene can be more accurately quantified by using the PCR amplification monitor method as shown in Example 8 in the process of gene amplification.
  • 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 (DNA or reverse transcript of RNA).
  • the detection target DNA or reverse transcript of RNA.
  • 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 is determined based on 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, Pro Natl. Acad. Sci.
  • the test for an allergic disease of the present invention may be performed by detecting the protein encoded by “787”.
  • a Western blotting method using an antibody that binds to a protein encoded by “787”, an immunoprecipitation method, an ELISA method, and the like can be used.
  • Antibodies to the protein encoded by the "787" of the present invention can be obtained as polyclonal antibodies or monoclonal antibodies using techniques well known to those skilled in the art (Milstein in C, et al., 1983, Nature 305 (5934): 537-40).
  • the protein or its partial peptide used as an antigen is prepared by incorporating the “787” gene or a part thereof into an expression vector, introducing this into an appropriate host cell, and preparing a transformant. It can be obtained by culturing the transformant to express the recombinant protein, and purifying the expressed recombinant protein from the culture or the culture supernatant.
  • a transformant obtained by culturing the transformant to express the recombinant protein, and purifying the expressed recombinant protein from the culture or the culture supernatant.
  • an allergen such as cedar pollen antigen.
  • 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 testing for allergic diseases.
  • the expression of the “787” gene expressed in T cells is reduced after pollen antigen exposure.
  • the “787” gene is a gene whose expression level decreases as a response of the living body to cedar pollen antigen stimulation, and screening of a therapeutic agent for hay fever can be performed by monitoring the expression of the “787” gene.
  • the expression level of the “787” gene is reduced by pollen antigen exposure in both healthy and hay fever patients. Presence or absence of hay fever symptoms is presumed to be due to the difference since the response of the "787J gene" to antigen stimulation. However, even in such cases, decreased expression of the "787” gene corresponds to increased T cell responsiveness Therefore, by monitoring the expression of the “7 87” gene, Can do it.
  • the method of the present invention for screening a candidate therapeutic compound for suppressing a T cell antigen-stimulated response 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 “787” is measured. I do.
  • lymphocytes are separated from peripheral blood, and the lymphocytes are stimulated with cedar pollen antigen or the like with in vitro. T cells are separated from the lymphocytes after the stimulation, and the transcript of the “787” gene is measured.
  • a compound that suppresses a decrease in the transcription level of the “787” gene compared to a control (when no candidate drug is administered) is selected.
  • the stimulation with the pollen antigen is performed for the purpose of eliciting an antigen-specific allergic reaction in T cells and determining the therapeutic effect of the candidate compound on it.
  • the term “suppressing the decrease in the transcription amount of the“ 787 ”gene” means that a higher level of transcription amount is maintained by contact with a candidate compound as compared to antigen-stimulated T cells.
  • the compound is a compound to be selected in the screening method of the present invention.
  • peripheral blood lymphocytes are collected from a human mouse or the like, and the peripheral blood lymphocytes are stimulated with in vitro with a cedar pollen antigen or the like.
  • Candidate compounds are added during in vitro stimulation.
  • the T cells are then separated from the stimulated peripheral blood lymphocytes and the transcript of “787” is measured.
  • a compound that suppresses a decrease in the transcription amount of the “787” gene compared to a control (when the candidate drug is not contacted) is selected.
  • the screening of candidate therapeutic compounds that suppress the antigen-stimulated response of T cells of the present invention can also be performed using established T cells.
  • established T cells such as Molt4 cells and Jurka t cells are stimulated in vitro with a lymphocyte stimulator. Lymphocyte sting Examples of the intense substance include calcium ionophore (A23187), PMA, and phytohemagglutinin (PHA).
  • Add candidate drug during invitro stimulation Thereafter, the transcription amount of the “787” gene in the established T cells is measured. As a result of this measurement, a compound that suppresses a decrease in the transcription amount of the “787” gene compared to a control (when the candidate drug is not contacted) is selected.
  • Detection of the expression of the "787" gene in the screening of a therapeutic drug candidate compound that suppresses the T cell antigen-stimulatory response according to the present invention can be performed by detecting the nucleic acid hybridizing to the "787" gene in the same manner as in the test for allergic disease of the present invention.
  • the hybridization can be carried out using a hybridization technique using DNA as a probe, or a gene amplification technique using 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.
  • a method utilizing the gene amplification technique an RT-PCR method can be used.
  • the expression of the “787” gene can be more accurately quantified by using a PCR amplification monitoring method as shown in Example 8 in the gene amplification process.
  • test compounds used in these screenings include compound preparations synthesized by existing chemical methods such as steroid derivatives, compound preparations synthesized by combinatorial chemistry, extracts of animal and plant tissues, or microbial cultures.
  • a mixture containing a plurality of compounds such as a product, a sample purified from the mixture, and the like.
  • the compound isolated by the method of the present invention for screening a candidate therapeutic compound for suppressing a T cell antigen-stimulated response is a candidate for a drug that suppresses an immune response.
  • 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. For example, it is administered to a patient together with a pharmacologically acceptable carrier or vehicle (such as saline, vegetable oils, suspensions, surfactants, stabilizers, etc.). Dosage depends on the nature of the compound Performed percutaneously, intranasally, transbronchially, intramuscularly, intravenously, or orally. 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. 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 values of cedar pollen-specific IgE antibodies in each blood sample of Test Subjects A to (sample numbers 1 to 18) were 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 diagram showing changes in the expression of “787” 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. 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 considered to be atopic predisposition groups (hereinafter also referred to as “patients”), and two subjects H and were considered to be healthy subjects (hereinafter also referred to as “normal groups”). Of the eight subjects with an atopic predisposition, seven exhibited symptoms of allergic rhinitis after pollen dispersal.
  • patients atopic predisposition groups
  • normal groups two subjects H and were considered to be 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 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 at 4 to collect 1 ml of the supernatant as plasma.
  • the lymphocyte fraction obtained in Example 2 was centrifuged at 1200 i "pm for 4 and 5 minutes, and suspended in BSA / PBS at 10 8 per 100 / xl. The volume became about 201. This was transferred to an Eppendorf tube (1.5 ml), to which the CD3 microbead solution was added, and then left for 30 minutes at 4 to 10 ° C (not placed on ice at this time). The treatment was performed as follows using Yuichi (MACS) (manufactured by Miltenyi Biotech Inc.).
  • 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, cells labeled with CD3 microbeads were applied to the column. The column was washed three times with 500 ⁇ 1 (B cell fraction). The column was removed from the separation unit and placed on a tube 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 trypan blue to count the number of cells.
  • the total cell number was about 4 ⁇ 10 6 .
  • RNA from T cells For preparation of total RNA from T cells, use RNeasy Mini (manufactured by Qiagen). Performed according to the attached manual. 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 i "pm for 5 minutes, and the supernatant was removed by aspirating. A 350 // 1 lysis buffer RLT (containing 2-mercaptoethanol) solution was added to the precipitate. At this stage, the lysate of the cells in the RLT buffer could be stored at -70 ° C. If the lysate of the cells had been stored frozen, 10-15 at 37 ° C.
  • lysate was homogenized with a syringe equipped with a 20 G force teran needle, and then purified with Kia Shredda ( (That is, the lysate of 3501 cells was usually applied to a Kyaschlets d'Aunit using a Pittman.
  • wash Buffer RPE containing ethanol
  • 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, DEPC-treated water 301 was applied, the lid was put on the tube, and the tube was allowed to stand for 10 minutes, followed by centrifugation at 11500 ⁇ m for 10 minutes to obtain total RNA. Measure and if the volume is low, The sample was placed in a new 1.5 ml tube, water 301 treated with DEPC was applied, the lid was put on the tube, the tube was set for 10 minutes, and centrifuged at 11,500 rpm for 10 minutes.
  • DNase treatment was performed to remove DNA from total RNA prepared from T cells. Reaction 2 Unit DNase (Nitsubon Gene) and 50 units RNase inhibitor Yuichi
  • DD Fluorescence differential display
  • 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 zg of RNA for each of the three anchor primers. All cDNAs were diluted to a final concentration equivalent to 0.4 ng / l RNA and used for experiments.
  • a DD-PCR reaction was performed using cDNA equivalent to 1 ng RNA per reaction. Table 1 shows the composition of the reaction solution.
  • 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 kept at 72 ° C for 5 minutes, and then continuously 4T :.
  • the primer pairs used were primers AG1 to 110 and AG111 to GT15A (SEQ ID NO: 2), GT15C (SEQ ID NO: 3), and GT15G (SEQ ID NO: 4), which are anchor primers, respectively.
  • a total of 287 reactions were performed by combining 199 and AG 200-287.
  • an oligomer composed of 10 nucleotides having a GC content of 50% was designed, synthesized, and used.
  • a 6% denaturing polyacrylamide gel was prepared, 2.5 1 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.
  • 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 “787” band is GT15G (SEQ ID NO: 4) as an anchor primer, DD analysis using AG274 (TGACCTAGCT / SEQ ID NO: 5) as an imaginary animal revealed that the expression of “787” was stronger in 4 out of 8 subjects before dispersal than after dispersal.
  • a gel containing the band of “787” 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 290 bp was amplified.
  • GT15G was used as the anchor primer, and AG274 was used as the optional primer.
  • the amplified DNA fragment was cloned into a plasmid vector pCR2.l (Invitrogen) to obtain a plasmid p787-5 having a DNA fragment of about 290 bp.
  • the nucleotide sequence of the DNA fragment was determined according to a conventional method.
  • the expression amount of "787" was quantified by the TaqMan method using ABI-PRISM7700. This method is a system for real-time quantitative detection of PCR-amplified DNA strands using fluorescent dyes.
  • blood samples before and after cedar pollen scattering were newly 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 total RNA samples.
  • primers 787-5 ′ AGCTTCTGGACAGCCCAGTC / SEQ ID NO: 6
  • 787-3 ′ GGAGTTAGCCAGGCAGCAAAZ sequence ID: 7
  • TaqMan probe 787-13SEQ TGGTGATGGCTGGAGGGAGTGATTGZ Column number: 8
  • the TaqMan probe 787-13SEQ was used by fluorescently labeling the 5 end with FAM (6-carboxyfluorescein) and the 3 end with TAMRA (6-carboxy-tetramethytri rhodamine).
  • FAM 6-carboxyfluorescein
  • TAMRA 6-carboxy-tetramethytri rhodamine
  • type II reverse transcribed cDNA from 44 total RNAs using poly T (12 to 18 mer) as a primer was used.
  • a serial dilution of the plasmid p787-5 obtained in Example 7 was used as a type II reaction.
  • Table 3 shows the composition of the reaction solution for monitoring PCR amplification.
  • Reaction composition of ABI-PRISM 7700 (reaction volume per 1 liter) Sterile distilled water 25.66 (L)
  • Table 4 shows the number (copy number) of “787” in each sample corrected for the copy number of / 3-actin. For the correction, the average copy of -actin in all samples was determined, and the copy number of "787" in each sample was divided by the relative value of / 3-actin in each sample when it was set to 1.
  • a primer was designed in the determined "787" sequence, and further upstream sequencing was attempted using a Marathon cDNA Amplification Kit (CLONTECH). Using cDNA from Human Leukemia K-562 cell line (CLONTECH), a cDNA for Marathon was prepared and type II. PCR was carried out using the primer 787_M1U (TTCACGCGGT CTCTTGTAMGTTZ SEQ ID NO: 11) designed in a known sequence region and the adapter primer attached to the kit.
  • the PCR reaction conditions were as follows: after heating at 94 ° C for 30 seconds, 5 cycles of ⁇ 945 seconds, 704 minutes '', 5 cycles of ⁇ 94 ° C for 5 seconds, 68 minutes for 4 minutes, '' ⁇ 94 ° C for 5 seconds, 66 4 minutes "for 25 cycles. As a result, an amplification product of about 1.3 kb was obtained. When this DNA sequence was determined, a 1305 bp sequence including the previously determined “787” sequence was obtained. This sequence is shown in SEQ ID NO: 1.
  • the attached AP2 Primer (ACTCACTATAGGGCTCGAGCGGCZ sequence number: 14) and the 787-specific 787-2R Primer (GCTCCCTAGTCTCTCTCACCTCCT sequence number: 15) was used to perform a PCR reaction.
  • the reaction conditions for the PCR were as follows: primary: one cycle of ⁇ 94 ° C for 3 minutes '', followed by ⁇ 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 3 minutes '' after 35 cycles of ⁇ 72 ° C One cycle of “C5 min” was followed by continuous 4 ° C.
  • the amplified DNA fragment was excised from the gel, cloned with plasmid vector PCR2.1 (Invitrogen), and a plasmid containing a DNA fragment of about 600 bp was cloned. Obtained.
  • plasmid vector PCR2.1 Invitrogen
  • the nucleotide sequence of the DNA fragment was determined according to a conventional method. As a result, about 200 bp of the upstream sequence could be obtained. The sequence is shown in SEQ ID NO: 16.
  • 5 'RACE analysis was performed using Marathon-Ready cDNA (CLONTECH) Bone Marrow as type II.
  • the attached API Primer CCATCCTAATACGACTC ACTATAGGGCZ SEQ ID NO: 12
  • 787-yRl Primer AGCCCTCT A PCR reaction was performed using GAATCTCCACTCTCTAZ SEQ ID NO: 17.
  • the attached AP2 Primer ACTCACTATAGGGCTCGAGCGGC / S2 column number: 14
  • the 787-specific 787-yR2 Primer TCCCTTACCAGATACTACCTCG TG / SEQ ID NO: A PCR reaction was performed using (18).
  • the 787-yRl Primer and 787-yR2 Primer were designed based on the nucleotide sequence obtained from "Recovery of band excised by 5 'RACE analysis and sequencing 1".
  • the reaction conditions for the PCR were as follows: primary: one cycle of ⁇ 94 ° C for 3 minutes '' followed by ⁇ 94 ° C for 30 seconds, 6 CTC for 30 seconds, 72 ° C for 1 minute '' after 35 cycles of ⁇ 72 ° C Min) for one cycle and then continuously at 4 ° C.
  • the second is one cycle of ⁇ 94 ° C for 3 minutes '' followed by one cycle of ⁇ 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for one minute '' followed by one cycle of ⁇ 72 ° C for 5 minutes '' Thereafter, the temperature was continuously raised to 4 ° C.
  • TaKaRa Ex Taq (TaKaRa) was used as a Taq enzyme, and reaction solutions were prepared from the attached reaction reagents according to the attached manual.
  • a 1.2% agarose gel was prepared, 5 ⁇ 1 sample was applied, and electrophoresis was performed at 100 V constant voltage for 30 minutes. Thereafter, electrophoretic images were obtained by UV transillumination.
  • the amplified DNA fragment was excised from the gel, cloned with the plasmid vector PCR2.1 (Invitrogen), and the plasmid containing the approximately 1.2 kbp DNA fragment was cloned. Obtained.
  • the nucleotide sequence of the DNA fragment was determined according to a conventional method. As a result, a further upstream sequence of about 1 kb was obtained. The sequence is shown in SEQ ID NO: 19. Based on these findings, the nucleotide sequence of “787” was finally 2565 bp. The nucleotide sequence of "787" is shown in SEQ ID NO: 20.
  • the present invention is used as an indicator of the response of T cells to antigen stimulation such as cedar pollen.
  • New genes have been provided. Using the expression of the gene of the present invention as an index, it has become possible to conduct a test for the presence or absence of a response of T cells to pollen antigen, or to screen a candidate therapeutic compound for suppressing the response of T cells to antigen stimulation.

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Abstract

La présente invention concerne un gène à expression fortement atténuée après dispersion du pollen. On a réussi à isoler ce gène en préparant des lymphocytes T des sujets avant et après la pollinisation, puis en observant le gène selon un procédé différentiel. Il s'est avéré que ce gène convient à l'étude des affections allergiques et à la recherche systématique de candidats composés de remèdes capables de réguler la réponse des lymphocytes T au stimulus d'un antigène.
PCT/JP2000/003192 1999-05-27 2000-05-18 Gene 787 associe a la pollinose Ceased WO2000073440A1 (fr)

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JP14878599 1999-05-27
JP11/148785 1999-05-27

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WO2000073440A1 true WO2000073440A1 (fr) 2000-12-07
WO2000073440A8 WO2000073440A8 (fr) 2001-03-15

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003083139A1 (fr) * 2002-04-03 2003-10-09 Genox Research, Inc. Procede de diagnostic de maladie allergique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BARUJ BENACERRAF ET AL.: "Histocompatibility-linked immune response genes", SCIENCE, vol. 175, 1972, pages 273 - 279, XP002930725 *
KAZUSHI HONDA: "Sugi kafunshou no meneki identeki kaiseki", ALLERGY NO RINSHOU, vol. 89, 1988, pages 111 - 115, XP002945874 *
KENICHI YAMAMURA ET AL.: "Functional expression of a micro-injected Ed(alpha) gene in C57BL/6 transgenic mice", NATURE, vol. 316, 1985, pages 67 - 69, XP002930724 *
SHOU MATSUSHITA: "Allergy shikkan no identeki koso", RINSHOU MENEKI, vol. 20, no. SUPPL. 13, 1988, pages 68 - 76, XP002945875 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003083139A1 (fr) * 2002-04-03 2003-10-09 Genox Research, Inc. Procede de diagnostic de maladie allergique

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