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WO2000065050A1 - Gene 795 associe a la pollinose - Google Patents

Gene 795 associe a la pollinose Download PDF

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Publication number
WO2000065050A1
WO2000065050A1 PCT/JP2000/002734 JP0002734W WO0065050A1 WO 2000065050 A1 WO2000065050 A1 WO 2000065050A1 JP 0002734 W JP0002734 W JP 0002734W WO 0065050 A1 WO0065050 A1 WO 0065050A1
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Prior art keywords
gene
seq
expression level
cells
nucleic acid
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PCT/JP2000/002734
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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
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 WO2000065050A1 publication Critical patent/WO2000065050A1/fr
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Ceased legal-status Critical Current

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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

  • 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 proposed a method 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 divided the subjects into a group having a high IgE value for cedar pollen (a group predisposed to cedar pollinosis) and other groups (normal subjects), and determined the expression level of the isolated “795” 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. Furthermore, the results of the analysis of the base sequence As a result, it is a non-splicing form of this viability S vimentin, and the expression pattern of vimentin is significantly lower in the cedar pollinosis predisposition group compared to healthy subjects as in ⁇ 795 '' It revealed that. For this reason, the present inventors have found that it is possible to perform an allergic disease test and a screening of a candidate drug for a therapeutic agent for allergic disease using the expression levels of these genes as indices.
  • the present invention relates to a gene exhibiting a decreased expression level in a person having an allergic predisposition, a method for testing an allergic disease using the expression level of the gene as an index, and a method for screening a candidate compound for a therapeutic drug for allergic disease. . More specifically, the present invention relates to the following nucleic acid molecule, and a test method or a screening method using the nucleic acid molecule as an index.
  • nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 24;
  • a nucleic acid molecule functionally equivalent to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 24, which is hybridized with a DNA consisting of the nucleotide sequence of SEQ ID NO: 24 under stringent conditions.
  • [3] A vector into which the nucleic acid molecule according to [1] or [2] has been inserted.
  • SEQ ID NO: 24 or the nucleotide sequence of SEQ ID NO: 25 in a biological sample A method for testing an allergic disease, comprising the step of measuring the expression level of a gene comprising the gene and comparing it with a control (in the case of a healthy subject).
  • the biological sample is blood, and the expression level of the gene is measured by measuring a protein comprising the amino acid sequence shown in SEQ ID NO: 26 and Z or a fragment thereof in blood. the method of.
  • 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) 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).
  • 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
  • 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 allergic diseases can include bronchial asthma, allergic rhinitis, atopic dermatitis, hay fever, or insect allergy.
  • Allergic diathesis 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” includes DNA and RNA.
  • the “test for allergic disease” in the present invention includes not only a test for a patient who has developed an allergic disease, but also a determination of whether or not a subject who has not developed an allergic disease has an allergic predisposition. Inspections are 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 “795” that is correlated with an IgE production response to cedar pollen of an individual.
  • the nucleotide sequence of the “795” cDNA found by the present inventors is shown in SEQ ID NO: See Figure 24.
  • “795” is based on the base sequence (SEQ ID NO: 1, 1384 bp) of the fragment isolated by the DD method, and is based on the RACE method (Frohman, MA et al .: Pro Natl. Acad. Sci. USA, 85 : 8992, 1988), and the nucleotide sequence at the 5 'side was determined, and finally the nucleotide sequence of 4467 bp (SEQ ID NO: 24) was determined.
  • the nucleotide sequence of “795” (SEQ ID NO: 24) in the present invention was confirmed by a homology search to be a non-slicing form containing exon 1 and exon 2 of vimentin, a known protein. .
  • Vimentin is a constituent protein of vimentin filament which belongs to the type III intermediate filament protein (Mo 1 ⁇ Cell. Biol. 6 / 11,3614-3620,1986). It is thought to form a network that spreads throughout the cytoplasm, dynamically integrating cells. Vimentin has also been confirmed to exist in undifferentiated cells and tumor cells. However, there is no report that vimentin is associated with an allergic disease, and the findings obtained by the present inventors are novel.
  • the nucleic acid molecule of the present invention can hybridize not only with the nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 24 but also under stringent conditions with the nucleic acid molecule, and a nucleic acid functionally equivalent to the nucleic acid molecule Including molecules.
  • the stringent condition may be, for example, the following condition. Hybridization conditions can be adjusted according to the chain length of the nucleic acid molecule and the constituent bases.
  • functionally equivalent to “795” means that expression is significantly lower in the atopic predisposing group (powder value for cedar pollen is 3.5 AU / ml or more) than in the non-atopic predisposing group. means. Since nucleic acid molecules that can hybridize under stringent conditions are nucleic acid molecules that are structurally highly homologous, screening under such conditions allows the “795” in the present invention to be screened. A functionally equivalent nucleic acid molecule can be obtained.
  • the nucleic acid molecule of the present invention can be obtained by a known method based on the nucleotide sequence information shown in SEQ ID NO: 24.
  • the vimentin gene can also be obtained by a known method based on the nucleotide sequence that has already been clarified.
  • the desired cDNA can be selected by screening a cDNA library of human T cells with a probe set based on the nucleotide sequence of SEQ ID NO: 24.
  • PCR can be performed using primers set based on the nucleotide sequence shown in SEQ ID NO: 24 to amplify the target gene.
  • a cDNA derived from a non-human animal lymphocyte is used as a screening library or template for PCR, homologues of other species of “795” can be obtained.
  • the nucleotide sequence shown in SEQ ID NO: 24 can also be chemically synthesized.
  • Such molecular genetic techniques include known methods (Sambrook), Fritsch, EF, and Maniatis T. Molecular cloning: A Laboratory Manual (2nd edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor. ) Can be used for ij.
  • the nucleic acid molecule or vimentin gene of the present invention thus obtained is useful as a diagnostic index for allergic disease or as an index for screening a therapeutic agent for allergic disease.
  • the present invention provides a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 24, Relates to a polynucleotide that hybridizes to its complementary strand, the polynucleotide having a chain length of at least 15 nucleotides.
  • a polynucleotide that can hybridize to a given base sequence not only those specifically hybridizing to the nucleotide sequence of SEQ ID NO: 24 but also methods for designing probes and primers capable of hybridizing to an unknown nucleotide sequence similar in structure are known.
  • the polynucleotide having the designed base sequence can be chemically synthesized by a technique such as the dideoxy method.
  • a polynucleotide comprising a required nucleotide sequence can be obtained by cleaving a 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.
  • “795” and vimentin genes 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, using the expression of the “795” gene (that is, including transcription into mRNA and translation into protein) as an index, it is possible to test for allergic diseases and to screen candidate compounds for therapeutic drugs for allergic diseases. Conceivable.
  • cedar pollinosis is particularly preferred as an allergic disease to be a control for examination and treatment.
  • the detection of the expression of the gene of “795” or vimentin in the test for allergic disease can be performed by using a hybridization technique using a nucleic acid that hybridizes to the gene of “795” or vimentin as a probe, or a method using these techniques. It can be carried out by using gene amplification technology using DNA that hybridizes to the gene as a primer.
  • a nucleic acid molecule which specifically hybridizes to “795” or vimentin gene and has a chain length of at least 15 nucleotides is used.
  • “specifically hybridizes” means, under ordinary hybridization conditions, preferably stringent hybridization. Under hybridization conditions, refers to no significant cross-hybridization of DNA and / or RNA encoding other genes.
  • an Express Hybridization Solution manufactured by CLONTECH
  • the probe and transfer membrane are hybridized with 68, and finally washed with 50% in a solution of 0.IX SSC, 0.05% SDS, and then washed with 50%. Can be a gentle condition.
  • 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, labeling by nick translation using DNA polymerase 1, terminal labeling using polynucleotide kinase, fill-in labeling using cleno-fragment (Berger SL, Kernel AR. (198 7) Guide to Molecular Cloning Techniques, Method in Enzymology, Academic
  • Testing for allergic diseases using the hybridization technology 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 level of “795” or vimentin gene can be more accurately quantified by using the PCR amplification monitor method as shown in Example 8 in the gene amplification process.
  • "795" or vimentin It is desirable to measure the expression level of each gene alone. By hybridizing a probe or a primer with a base sequence specifically found in any of the genes as a target, the expression level of each gene can be clarified individually. Alternatively, it is possible to separately measure the expression levels of both using the region found in both, such as exon 1 and exon 2 of the vimentin gene.
  • the base sequence corresponding to the intron does not exist in the transcript that has undergone splicing of the vimentin gene, even if the same region is used as the target base sequence of the primer, the length of the generated amplification product differs. coming out. Further, by setting a probe for the PCR amplification monitoring method in the intron region, the amplification of “795” can be specifically followed. If a common nucleotide sequence is used as a target, the expression level of both can be determined comprehensively.
  • probes that are labeled with different fluorochromes 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 separate and the fluorescence is detected. This fluorescence is detected in real time.
  • the number of copies of the target in the target sample is determined by the number of linear cycles of PCR amplification by simultaneously measuring a standard sample whose copy number is known for the target (Holland, PM et al., 1991). Natl. Acad. Sci.
  • the test for an allergic disease of the present invention may be performed by detecting a vimentin protein.
  • the amino acid sequence of vimentin is known (SEQ ID NO: No .: 26).
  • a Western blotting method using an antibody that binds to a vimentin protein an immunoprecipitation method, an ELISA method, or the like can be used.
  • a lysate of T cells a blood sample such as serum or plasma can be used as a sample for protein detection.
  • a whole blood sample containing T cells can be lysed into a sample.
  • the antibody of the vimentin protein in the present invention can be obtained as a polyclonal antibody or a monoclonal antibody using techniques well known to those skilled in the art (Milstein C, eta, 1983, Nature 305 (5934): 537-40). ).
  • a vimentin gene SEQ ID NO: 25
  • a part thereof is inserted into an expression vector, and this is introduced into an appropriate host cell to prepare a transformant. It can be obtained by culturing the transformant to express a recombinant protein, and purifying the expressed recombinant protein from a culture or a culture supernatant.
  • the present invention relates to a polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 24 or SEQ ID NO: 25, or a nucleotide sequence complementary to a complementary strand thereof, and having a chain length of at least 15 nucleotides.
  • the polynucleotide of the present invention is used as a probe or a primer for detecting a gene used as an index for a test.
  • the expression of “795” or vimentin gene expressed in T cells is decreased in the group of hay fever patients with high IgE specific to pollen antigen. Even in allergic patients who show responsiveness to an antigen other than cedar pollen, expression of ⁇ 795 '' or vimentin gene is reduced while T cell responsiveness to the antigen is enhanced. May be In such cases, decreased expression of these genes corresponds to increased T-cell responsiveness, and thus monitoring of expression of the “795” or vimentin gene will increase the risk of allergic disease. Can be performed. That is, the present invention relates to the use of the polynucleotide in a method for screening a candidate compound for a therapeutic drug for an allergic disease.
  • 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 ⁇ 795 '' or vimentin gene The transcript of is measured.
  • lymphocytes are separated from peripheral blood, and the lymphocytes are stimulated in vitro with cedar pollen antigen or the like. T cells are separated from the lymphocytes after the stimulation, and their “795” or viment in gene transcript is measured.
  • “795” or a compound that increases the transcription amount of the vimentin gene 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.
  • peripheral blood lymphocytes are collected from hay fever humans or mice 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 the transcript of “795” or vimentin gene is measured. As a result of this measurement, “795” or a compound that increases the transcription amount of the vimentin 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 Mol t4 cells and Jurkat cells are stimulated in vitro with a lymphocyte stimulator.
  • lymphocyte stimulators include calcium ionophore (A23187), PMA, and phytohemagglutinin (PHA). And so on.
  • lymphocyte stimulators include calcium ionophore (A23187), PMA, and phytohemagglutinin (PHA). And so on.
  • Candidate drugs are added during in vitro stimulation. Thereafter, the transcription amount of “795” or vimentin gene in the established T cells is measured. As a result of this measurement, “795” or a compound that increases the transcription of the vimentin gene is selected.
  • Detection of the gene expression of "795" or vimentin in the screening of candidate compounds for the treatment of allergic diseases is carried out by hybridization using a nucleic acid that hybridizes to "795" or vimentin gene as in the test for allergic diseases of the present invention. It can be carried out by using a technique 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 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 “795” gene can be more accurately quantified by using a PCR amplification monitoring 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, compound preparations synthesized by combinatorial chemistry, and extracts of animal and plant tissues or Examples thereof include a mixture containing a plurality of compounds such as a microorganism culture, and a sample purified therefrom.
  • 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.
  • pharmacologically acceptable carriers or vehicles saline, vegetable oils, suspensions, (Activators, 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 values of cedar pollen-specific IgE antibodies in each blood sample of subjects A to ⁇ 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 graph showing changes in the expression of “795” 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 shows the results of measuring the expression level of “795” in hay fever patients. The vertical axis indicates the copy number (copy / ng RNA) of “795”, and the horizontal axis indicates the sample number.
  • FIG. 4 shows the results of measuring the expression level of vimentin in hay fever patients.
  • the vertical axis shows the copy number (copy / ng RNA) of “795”, and the horizontal axis shows the sample number.
  • FIG. 5 is a diagram showing changes in the expression of vimentin in the high IgE group and the normal IgE group when grouped according to cedar pollen-specific IgE values. 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 regarded as an atopic predisposition group (hereinafter also referred to as “patient”), and two 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
  • ⁇ cells from 10 ml of blood proceed 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). 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 for 4 and 5 minutes, and suspended in BSA / PBS at 10 8 per 100 ⁇ 1. The volume was about 20 l. 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 by Magnetic Celso Ichiichi (MACS) (Miltenyi Biotech Inc.) as follows.
  • MCS Magnetic Celso Ichiichi
  • the MS + / RS + column was attached to a Mini MACS or Vario MACS separation unit (without needles). 500 / xl BSA / PBS was gently applied to the column and the buffer was poured. Next, apply the cells labeled with CD3 microbeads to the column. I did it. The column was washed three times with (cell fraction). The column was removed from the separation unit and placed on a tube for collecting the eluate. 1 ml of BSA / PBS was applied to the column, and positive cells were rapidly flushed out using a plunger attached to the column. This was used as the T cell fraction.
  • the obtained T cell fraction was centrifuged at 1200 rpm for 5 minutes at 4.
  • 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. Total cell number was approximately 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. Lysis buffer-RLT was supplemented with 101 / ml 2-mercaptoethanol. The cell suspension was centrifuged at 1000-1200 ⁇ ⁇ ⁇ for 5 minutes, and the supernatant was removed by aspiration. 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 manufactured by Qiagen
  • the cell lysate had been stored frozen, incubate at 37 for 10-15 minutes, and if insolubles were visible, centrifuge for 3 minutes at maximum speed to collect only the supernatant.
  • the lysate was homogenized with a syringe equipped with a 20 G force teran needle and then treated with Q IAshredder. (That is, usually, 350 1 cell lysate was applied to the Kyaschlets Danitto using a Pitman. This was centrifuged at 1500 rpm for 2 minutes, and the effluent was collected.) 350 il of 70% ethanol was added. Mix well by pipetting.
  • An RNeasy spin column was attached to the attached 2 ml tube, a lysate mixture of cells was applied, centrifuged at 8000 Xg (11500 rpm) for 1 minute, and the effluent was discarded.
  • Posh buffer RW1700 was applied to the column, and the tube was capped for 5 minutes. The mixture was centrifuged at 11,500 rpm for 15 seconds, and the effluent was discarded.
  • Attach the column to a new 2 ml tube and wash with Pash Buffer RPE (containing ethanol) 500 1 was applied to the column, and centrifuged at 11500 15m for 15 seconds, and the effluent was discarded.
  • the wash buffer RPE5001 was applied to the column and centrifuged at maximum speed for 2 minutes.
  • the column was mounted in a new 1.5 ml tube, DEPC-treated water 301 was applied, and the lid was capped and allowed to stand for 10 minutes. Centrifugation was performed at 11500 ⁇ ⁇ ⁇ for 10 minutes to obtain total RNA. Measure the concentration, and if the volume is low, replace the column with a new one and place it in a 5 ml tube. Centrifuge.
  • DNase treatment was performed to remove DNA from total RNA prepared from T cells.
  • Fluorescent differential display F1 uorescent Diferential Display, abbreviated as “DD”) using total RNA prepared from T cells Analysis of the literature (T. I to et al., 1994, FEBS Let t. 351: 231-236).
  • Total RNA prepared from T cells was reverse transcribed to obtain cDNA.
  • Three anchors for the primary DD-PCR reaction CDNA was prepared using 0.2 g each of total RNA for each of the primers.
  • cDNA was prepared using 0.4 g of RNA for each of the three anchor primers. All cDNAs were diluted to a final concentration of 0.4 ng / ⁇ l RNA and used in the 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.
  • PCR reaction conditions were as follows: 3 cycles at 95, 5 minutes at 40, 5 minutes at 72, 1 cycle, followed by 30 cycles of 94 ⁇ 5 seconds, 2 minutes at 40, 1 minute at 72. 5 min at 72, then continuously at 4
  • the primer pair used is an anchor primer: GT15A (SEQ ID NO: 2), GT15C (SEQ ID NO: 3), and GT15G (SEQ ID NO: 4).
  • a total of 287 reactions were performed by combining 199 and AG 200-287.
  • As an optional primer use 10 nucleosides with a GC content of 50%. Oligomers composed of tides were 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 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 “795” was found by DD analysis using GT15A (SEQ ID NO: 2) as an anchor primer and AG109 (TGTCACGGTT SEQ ID NO: 5) as an arbitrary primer.
  • the gel containing the “795” band was cut out, stored in a TE solution, and heated for 6 minutes (TC, 10 minutes) to elute the DNA from the gel.
  • PCR was performed under the same conditions as DD-PCR for type I, and a DNA fragment of about 180 bp was amplified using GT15A as an anchor primer and AG109 as an optional primer. Invitrogen) was used to obtain a plasmid p795-50 carrying a DNA fragment of about 180 bp.
  • An EST sequence (N62037) homologous to 795 was extracted from dbEST, and then extracted using ABI AutoAssembler.
  • Example 8 Quantification by ABI-7700 The expression amount of “795” was quantified by the TaqMan method using ABI-PRISM7700. 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.
  • the TaqMan probe T795 was used with its 5 ′ end labeled with FAM (6-carboxyfluorescein) and its 3 ′ end with TARA (6-carboxy-tetramethytri rhodamine).
  • FAM 6-carboxyfluorescein
  • TARA 6-carboxy-tetramethytri rhodamine
  • type III cDNA obtained by reverse transcription of poly T (12 to 18 mer) as a primer from 44 kinds of total RNA was used. The reaction was carried out using a serial dilution of plasmid p795-50 obtained in Example 7 for the standard curve for calculating the copy number. Table 3 shows the composition of the reaction mixture for monitoring PCR amplification.
  • Table 4 shows the number (copy number) of “795” in each sample corrected for the copy number of / 3-actin. For the correction, the average copy of -actin in all samples was obtained, and the copy number of "795" in each sample was divided by the relative value of 0-actin in each sample when it was set to 1.
  • two-way analysis of variance was performed.
  • 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).
  • 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. Tests for two-way analysis of variance were performed using StatView software (Abacuus Concepts, Inc.).
  • PCR reaction For the second (nested) 5 'RACE-PCR reaction, the attached AP2 Primer (SEQ ID NO: 12: ACTCACTATAGGGCTCGA) A PCR reaction was performed using (GCGGC) and 795- 5R-8 Primer (SEQ ID NO: 13: GAGAGTCTACAAAC CTGTCTGA) specific within “795”.
  • the PCR reaction conditions were as follows: primary: one cycle of “94 at 9 minutes” followed by 43 cycles of “94 at 30 seconds, 55 at 30 seconds, 72 at 3 minutes” After that, one cycle of “5 minutes at 72” was performed for one cycle, and then continuously at 4 minutes.
  • the second is “94 for 3 minutes” for one cycle, followed by “94 for 30 seconds, 60 for 30 seconds, and 72 for 2 minutes” after 15 cycles, and then “72 for 5 minutes. For one cycle and then continuously for four.
  • the first reaction uses AmpliTaq Gold (PERKIN ELMER) and the second reaction uses TaKaRa Ex Taq (TaKaRa), and the reaction solution is prepared from the attached reagent according to the attached manual. Was prepared.
  • the amplified DNA fragment was excised from the gel, cloned into a plasmid vector pCR2.1 (Invitrogen), and a plasmid containing a DNA fragment of about 600 bp was cloned. Obtained.
  • a plasmid DNA the nucleotide sequence of the DNA fragment was determined according to a conventional method. As a result, a further upstream sequence of about 500 bp was obtained. The sequence is shown in SEQ ID NO: 14.
  • RACE analysis was performed using Cap Site cDNA (NIPPON GENE) Human HeLa Cell as type III.
  • Cap Site cDNA NIPPON GENE
  • NIPPON GENE Cap Site cDNA
  • For the primary 5'RACE-PCR reaction use the attached IRC Primer (SEQ ID NO: 15: CAA GGTACGCCACAGCGTATG) t 795-yRl Primer (SEQ ID NO: 16: CG GCAGGTTTCAGCGGGACTTC) specific to "795" was used to perform a PCR reaction.
  • 795-yR2 Primer specific to “795” SEQ ID NO: 18
  • 795-yRl Primer and 795-yR2 Primer are ⁇ 5 'RACE It is designed based on the base sequence obtained from “Recovering and Sequencing Bands Excised by Analysis 1”.
  • the primary PCR reaction conditions were as follows: 1 cycle of 95 minutes for 1 cycle, followed by 35 cycles of 94 seconds for 30 seconds, 60 seconds for 30 seconds, and 72 for 1 minute.
  • TaKaRa Taq (TaKaRa) was used as a Taq enzyme, and glycerol was added to the attached reaction reagent to a final concentration of 5% to prepare reaction solutions.
  • a 1.2% agarose gel was prepared, a 5 / ⁇ sample was applied, and electrophoresed at 100 V constant voltage for 30 minutes. Thereafter, electrophoretic images were obtained by UV transillumination overnight.
  • 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 1 kbp I got Using a plasmid DNA, the nucleotide sequence of the DNA fragment was determined according to a conventional method. As a result, about 800 bp of the upstream sequence could be obtained.
  • the sequence is shown in SEQ ID NO: 19. From the NCBI BLAST Search, the nucleotide sequence from No. 1 to No. 705 of the sequence shown in SEQ ID NO: 19 is the same as the nucleotide sequence from No. 147 to No.
  • Example 12 As a result of Example 12, it was determined that “795” was a non-splicing form of vimentin. Therefore, the expression amount of “vimentin” was quantified by the TaqMan method using ABI-PRISM7700. As a sample, a total of 44 kinds of total RNA samples as in Example 8 were used to quantify the expression amount of the vimentin gene.
  • PCR amplification quantification method was performed based on the sequence of the vimemt in gene (GenBank accession number: M14144).
  • PCR was performed using peripheral blood Tcell-derived cDNA as type III primer-vimentin2F (GACATTGAGATTGCCACCTACAGZ SEQ ID NO: 20) t vimentinR (GGGTATCAA CCAGAGGGAGTGM / SEQ ID NO: 21).
  • the obtained and amplified 130 bp DNA fragment (SEQ ID NO: 23) was cloned into a plasmid vector (pGEM, Promega).
  • PCR amplification and quantification were performed using vimentinTQ (TCCCTGAACCTGAGGGAAACT MTCTGGA / SEQ ID NO: 22) as a TaqMan probe. Other conditions were the same as in Example 8.
  • Figures 7 and 4 show the numbers (copy numbers) of “795” and vimentin mRNA in each sample, corrected for the copy number of 3-actin. For the correction, the average copy of / 3-actin in all samples was determined, and the copy number of vimentin in each sample was divided by the relative value of -actin in each sample when it was set to 1. In addition, the vimentin mRNA copy numbers before and after pollen scattering were compared for each sample (Table 6).
  • 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 it has an allergic predisposition and a screening for candidate compounds for the treatment of 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/JP2000/002734 1999-04-27 2000-04-26 Gene 795 associe a la pollinose Ceased WO2000065050A1 (fr)

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JP12049499 1999-04-27

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Citations (1)

* 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

Patent Citations (1)

* 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

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
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 *
HONORE B. ET AL.: "Nucleotide sequence of cDNA covering the complete coding part of the human vimentin gene", NUCLEIC ACIDS RESEARCH, vol. 18, no. 22, 25 November 1990 (1990-11-25), pages 6692, XP002930137 *
KUZUSHI HONDA: "Sugi kafun sho no meneki idengaku teki kaiseki HLA to rensa shita sugi kafun kogen ni taisuru meneki yokusei idenshi no shoumei to to kaiseki", FUKUOKA IGAKU ZASSHI, vol. 80, no. 1, 25 January 1989 (1989-01-25), pages 28 - 37, XP002946664 *
MITSURU MUNAKATA: "beta-Adrenalin juyotai idenshi", MITSURU MUNAKATA, 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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