WO2002075304A1 - Methode d'analyse d'une maladie allergique - Google Patents
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- WO2002075304A1 WO2002075304A1 PCT/JP2002/001916 JP0201916W WO02075304A1 WO 2002075304 A1 WO2002075304 A1 WO 2002075304A1 JP 0201916 W JP0201916 W JP 0201916W WO 02075304 A1 WO02075304 A1 WO 02075304A1
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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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
-
- 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
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- 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
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- 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 method for testing an allergic disease.
- Allergic diseases such as atopic dermatitis are considered to be multifactorial diseases. These diseases are caused by the interaction of the expression of many different genes, and the expression of these individual genes is affected by multiple environmental factors. For this reason, it is very difficult to elucidate the specific gene that causes a specific disease.
- allergic diseases are thought to be related to the expression of genes with mutations or defects, or to overexpression or decreased expression of specific genes.
- test methods have been attempted to prove the involvement of allergic reactions regardless of the allergen.
- a high serum IgE level may indicate that the patient has an allergic reaction.
- the serum IgE value is information corresponding to the total amount of allergen-specific IgE. It is easy to determine the total amount of IgE regardless of the type of allergen, but patients with diseases such as non-atopic bronchitis asthma may have low levels of IgE.
- the present invention provides a new index capable of testing an allergic disease, a method for testing an allergic disease based on the index, and a method for screening a compound for treating an allergic disease. Providing is an issue.
- the present inventors have conducted intensive research to solve the above-mentioned problems, and as a result, By isolating genes whose expression levels are different between diseased patients and healthy individuals and clarifying the relationship with allergic reactions, new targets for the treatment of allergic diseases can be found Thought.
- the present inventors searched for genes having different expression states between atopic dermatitis patients receiving steroid ointment treatment and healthy persons.
- the response to steroid treatment is altered expression of the dalcocorticoid receptor.
- the fluctuation of the gene expression between the patient and the healthy subject was not attributable to the steroid treatment itself. Conceivable.
- peripheral blood mononuclear cells were selected as biological samples for comparing gene expression states.
- peripheral blood mononuclear cells are closely related to alenoregism.
- genes whose expression fluctuates in peripheral blood mononuclear cells can be said to be genes closely related to allergic reactions.
- peripheral blood mononuclear cells are easy to obtain, a high yield to eliminate false negatives can be expected, and thus it is considered that the expression of a small amount of genes can be detected.
- the present inventors analyzed the expression state of the gene in peripheral blood mononuclear cells, and found that the expression was increased in mononuclear cells of patients with Matk ⁇ .megakaryocyte associated tyrosine kinase) I confirmed that.
- the Matk gene is a known gene.
- the protein encoded by the Matk gene has been known to be highly expressed in hematopoietic cell systems and brain, especially in breast cancer cells (Zrihan-Licht S. et al., J. Biol. Chem. 272: 1856-1863, 1997).
- the present inventors have made it possible to diagnose allergic diseases by using the expression level of the Matk gene or the activity of the protein encoded by the gene as an index, and The present inventors have found that screening of therapeutic drugs is possible, and completed the present invention.
- the present invention relates to the provision of a new index capable of detecting an allergic disease, a method for testing an allergic disease based on the index, and a method for screening a candidate compound for a therapeutic drug for an allergic disease. , And more specifically,
- a method for testing allergic diseases including the following steps:
- a reagent for detecting an allergic disease comprising a polynucleotide containing the base sequence of the Matk gene, or an oligonucleotide having a length of at least 15 bases having a complementary base sequence to its complementary strand,
- a reagent for testing allergic diseases comprising an antibody that recognizes a peptide containing the amino acid sequence of Matk protein;
- a method for screening a therapeutic drug for an allergic disease comprising the following steps:
- Allergy including a polynucleotide containing the nucleotide sequence of the Matk gene, or an oligonucleotide having a nucleotide sequence complementary to the complementary strand thereof and having a length of at least 15 nucleotides, and a cell expressing the Matk gene Kits for screening compounds for the treatment of diseases,
- an antibody recognizing a peptide containing an amino acid sequence of Matk protein and a kit for screening a candidate drug for the treatment of an allergic disease, comprising a cell expressing a Matk gene,
- An allergic disease model animal consisting of a transgenic non-human vertebrate with an increased expression level of a marker gene in mononuclear cells, wherein the marker gene is Matk or a gene functionally equivalent to Matk A model animal,
- a method for screening a therapeutic agent for an allergic disease comprising the following steps, wherein the gene is a gene functionally equivalent to the indicator gene power atk or Matk,
- a method for screening a remedy for allergic 'I' live disease comprising the following steps:
- a method for screening a remedy for allergic diseases comprising the following steps:
- a therapeutic agent for allergic diseases comprising as an active ingredient a compound obtainable by the screening method according to any one of [7], [12], [13], and [14]. ,
- Therapeutic agent for allergic diseases which contains Matk gene or a part of antisense DNA as an active ingredient
- a therapeutic drug for allergic diseases comprising as an active ingredient an antibody that recognizes a peptide containing the amino acid sequence of Matk protein,
- the present invention relates to a method for treating an allergic disease, comprising a step of administering a compound obtainable by the screening method according to any one of [7], [12], [13], and [14].
- the present invention also provides [7], [12],
- the present invention relates to a method for treating an allergic disease, comprising a step of administering the following component (a) or (b).
- the present invention relates to the use of the following component (a) or (b) in the manufacture of a pharmaceutical composition for treating an allergic disease.
- an allergic disease is a general term for diseases associated with allergic reactions. More specifically, allergens have been identified and Can be defined as having a deep link between exposure to cancer and the development of a lesion, and the lesion has demonstrated an immunological mechanism.
- the immunological mechanism means that white blood cells show an immune response by stimulation of allergen. Examples of allergens include mite antigens and pollen antigens.
- allergic diseases can include bronchial asthma, allergic rhinitis, atopic dermatitis, hay fever, or insect allergy.
- Allergic diathesis is a genetic factor transmitted from a parent with an allergic disease to a child.
- a familial allergic disease is also called atopic disease, and the genetic factors that cause it are predisposed to atopy.
- Atopic dermatitis is a generic term given to atopic diseases, especially those associated with skin inflammatory conditions.
- the method for testing for an allergic disease of the present invention includes a step of measuring the expression level of the Matk gene in a biological sample of a subject and comparing the measured level with the measured value of a healthy subject. As a result of comparison between the two cases, if the expression is higher than that in a healthy subject, the subject is determined to have an allergic disease.
- a standard value is usually set based on, for example, the expression level of the indicator gene in a healthy subject. Based on this standard value, for example, the range of 2 S.D. Techniques for setting a standard value and an allowable range based on a measured value of an indicator gene are known. If the expression level of the indicator gene in the subject is higher than the allowable range, the subject is determined to have an allergic disease. If the expression level is within the allowable range, the possibility of having an allergic disease is expected to be low.
- the expression level of the Matk gene includes the transcription of the gene into mRNA and the translation into protein. Therefore, the method of testing for allergic disease according to the present invention is performed based on the comparison of the expression intensity of the mRNA corresponding to the Matk gene or the expression level of the protein encoded by the gene.
- the determination can be performed according to a known gene analysis method. Specifically, for example, a hybridization technique using a nucleic acid that hybridizes to the gene as a probe or a gene amplification technique using a DNA that hybridizes to the gene of the present invention as a primer can be used.
- the probe or primer used in the test of the present invention can be set based on the base sequence of the Matk gene.
- the base sequence of the Matk gene and the amino acid sequence encoded by the gene are known.
- the nucleotide sequence of the Matk gene of the present invention is GenBank accession number: AC005777.
- the nucleotide sequence of the Matk gene is shown in SEQ ID NO: 11, and the amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO: 12.
- genes in higher animals are frequently associated with polymorphisms. Also, there are many molecules that produce isoforms consisting of mutually different amino acid sequences in the process of splicing. Even if the genes differ in nucleotide sequence depending on the polymorphic isoform, all genes involved in allergy that have the same activity as the Matk gene are included in the Matk gene of the present invention.
- the Matk gene includes not only humans but also homologs of other species. Therefore, the Matk gene in a species other than human refers to a homologue of the Matk gene specific to the species or an exogenous Matk gene introduced into the individual, unless otherwise specified.
- the homolog of the human Matk gene refers to a gene derived from a species other than human that can hybridize under stringent conditions using the human Matk gene as a probe.
- Stringent conditions generally indicate the following conditions. That is, hybridization is performed at 4 ⁇ SSC at 65 ° C., and washing is performed at 65 ° C. for 1 hour using 0.1 XSSC.
- the temperature conditions for hybridization and washing which greatly affect the stringency, can be adjusted according to the melting temperature (Tm). Tm is the ratio of the constituent bases in the hybridizing base pairs, Hybridization 1. Varies depending on the composition of the solution (salt concentration, sodium concentration of formamide-dodecyl sulfate). Therefore, those skilled in the art can experimentally or empirically set conditions that give equivalent stringency in consideration of these conditions.
- a polynucleotide consisting of the base sequence of the Matk gene or a polynucleotide containing at least 15 nucleotides complementary to a complementary strand thereof can be used.
- the "complementary strand” refers to one strand of a double-stranded DNA consisting of A: T (U in the case of a thigh) and G: C base pairs with respect to the other strand.
- the term “complementary” is not limited to a case where the sequence is completely complementary to at least 15 contiguous nucleotide regions, but is at least 70%, preferably at least 80%, more preferably 90 °, and still more preferably 95%. It is only necessary to have the above homology on the base sequence.
- the homology of the nucleotide sequences can be determined by an algorithm such as BLAST.
- Such a polynucleotide can be used as a probe for detecting the Matk gene and as a primer for amplifying the Matk gene.
- a primer When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp.
- probes at least a portion or the whole sequence of a Matk gene (or its complementary strand), DNA chain length of at least 15b P is used.
- the 3 ′ region must be complementary, but a restriction enzyme recognition sequence tag or the like can be added to the 5 ′ region.
- polynucleotide in the present invention can be DNA or RNA. These polynucleotides may be synthetic or natural.
- a labeled DNA is usually used as a probe DNA used for hybridization. Examples of the labeling method include the following methods.
- the term oligonucleotide means a polynucleotide having a relatively low degree of polymerization. Oligonucleotides are included in polynucleotides.
- Testing for allergic monogenic disease using hybridization techniques can be performed using, for example, Northern hybridization, dot blotting, a method using a DNA microarray, and the like. Furthermore, gene amplification techniques such as the RT-PCR method can be used. In the RT-PCR method, more quantitative analysis can be performed on the expression of the gene of the present invention by using the PCR amplification monitoring method in the gene amplification process.
- probes to be detected are hybridized using probes labeled on both ends with different fluorescent dyes that cancel each other's fluorescence.
- the 5, q-3, exonuclease activity of Taq polymerase causes the probe to become: ⁇ ! ⁇ , which separates the two fluorescent dyes and allows fluorescence to be detected. This fluorescence is detected in real time.
- the number of copies of the target in the target sample is determined by the number of linear cycles of PCR amplification by simultaneously measuring a standard sample with a clear copy number for the target (Holland, PM et al., 1991). Natl. Acad. Sci.
- the method for testing an allergic disease of the present invention can also be carried out by detecting a protein encoded by the Matk gene.
- a test method for example, a Western plotting method using an antibody that binds to the Matk protein, an immunoprecipitation method, an ELISA method, and the like can be used.
- Antibodies that bind to the Matk protein used for this detection can be obtained using techniques well known to those skilled in the art.
- the antibody used in the present invention can be a polyclonal antibody or a monoclonal antibody (Milstein C, et al., 1983, Nature 305 (5934): 537-40).
- a polyclonal antibody against the Matk protein is obtained by extracting blood from a mammal sensitized with the antigen and separating serum from the blood by a known method. Serum containing the polyclonal antibody can be used as the polyclonal antibody.
- a fraction containing a polyclonal antibody can be further isolated from the serum.
- immune cells are removed from a mammal sensitized with the above antigen, and are fused with myeloma cells and the like.
- the hybridoma thus obtained can be cloned, and the antibody can be recovered from the culture to obtain a monoclonal antibody.
- these antibodies may be appropriately labeled and used. Further, without labeling the antibody, a substance that specifically binds to the antibody, for example, protein A peptide G can be labeled and detected indirectly. As a specific detection method, for example, an ELISA method can be mentioned.
- a protein or a partial peptide thereof used as an antigen is prepared by incorporating a Matk gene or a part thereof into an expression vector, introducing this into an appropriate host cell, preparing a transformant, and culturing the transformant.
- Expression of recombinant protein It can be obtained by purifying the recombinant protein from a culture or a culture supernatant.
- an oligonucleotide comprising an amino acid sequence encoded by the gene or a partial amino acid sequence of an amino acid sequence encoded by a full-length cDNA can be chemically synthesized and used as an immunogen.
- an allergic disease can be tested using not only the expression level of the Matk gene but also the activity of the Matk protein in a biological sample as an index.
- Matk protein activity refers to the biological activity of the protein.
- the activity of the Matk protein can be detected based on a known method such as an ELISA method using an antibody against the Matk protein or a Western plot method.
- a biological sample of a subject is usually used as a sample.
- Blood, sputum, nasal mucosal secretions, and the like can be used as a biological sample, but peripheral blood mononuclear cells are preferably used.
- a method for collecting mononuclear cells from peripheral blood or the like is known.
- Mononuclear cells isolated from peripheral blood are particularly referred to as peripheral blood mononuclear cells (PBMC).
- PBMC peripheral blood mononuclear cells
- Mononuclear cells can be easily collected, for example, from heparin-collected blood by specific gravity centrifugation.
- Monocytes are a population of cells that include monocytes and lymphocytes. The use of a large amount of mononuclear cells in peripheral blood facilitates sample collection.
- Peripheral blood mononuclear cells can be prepared from peripheral blood by a known method. For example, it can be prepared by the method described in Example 1. If the prepared mononuclear cells are destroyed into a lysate, it can be used as a sample for immunological measurement of Matk protein. Further, the method for collecting a material other than the mononuclear cell of the present invention is also known.
- a lysate is prepared from the above biological sample, it can be used as a sample for immunological measurement of Matk protein.
- mRNA is extracted from this lysate, it can be used as a sample for measuring mRNA corresponding to the Matk gene.
- the indicator protein is secreted into the blood, the expression level of the gene encoding it can be compared by measuring the amount of the target protein contained in a body fluid sample such as blood or serum of the subject. It is possible.
- the above sample can be diluted with a buffer or the like, if necessary, and used in the method of the present invention.
- the measured value of the expression level of the Matk gene in the present invention can be corrected by a known method. With the correction, changes in the expression level of the gene in the cells can be compared.
- the measurement value is corrected based on the expression level of the Matk gene in each of the cells in the biological sample based on the measurement value of the expression level of a gene whose expression level does not fluctuate significantly (for example, a house key ⁇ -ping gene). This is done by correcting the measured values of Examples of genes whose expression levels do not fluctuate significantly include ⁇ -actin, GAPDH and the like.
- the Matk gene in the present invention showed an increased expression level in mononuclear cells of patients in comparison between healthy subjects and patients with atopic dermatitis. Therefore, an allergic disease can be tested using the expression level of the Matk gene as an index.
- the test for allergic disease in the present invention includes, for example, the following tests. Even if a patient cannot show an allergic disease by a general test while showing symptoms of suspected allergy, the test according to the present invention can easily determine whether or not he / she is a patient with an allergic disease. can do. More specifically, an increase in the expression of the Matk gene in a patient exhibiting a condition suspected of having an allergic disease indicates that the cause of the symptom is likely to be an allergic disease.
- tests can be done to determine if allergic symptoms are improving. In other words, it is useful for determining the therapeutic effect on allergic diseases. Elevated expression of the Matk gene in patients diagnosed with an allergic disease also indicates that the allergic disease is likely to be more advanced.
- the present invention also relates to a transgenic non-human animal model animal for allergic disease in which the expression level of the Matk gene in mononuclear cells is increased.
- the present invention has revealed that the expression level of the Matk gene in mononuclear cells is increased in mononuclear cells of patients with atopic dermatitis. Therefore, animals in which the expression level of the Matk gene has been artificially enhanced in mononuclear cells can be used as an animal model for allergic disease.
- the animal model for allergic disease is useful for elucidating in vivo changes in allergy. Furthermore, it is of great significance to elucidate the further function of the Matk gene by using the allergic disease model animal and to evaluate drugs that target the gene.
- the animal model for allergic disease according to the present invention is useful for elucidating the mechanism of allergic disease and for testing the safety of screened compounds. For example, if the animal model for allergic disease according to the present invention develops dermatitis or shows a change in a measurement value related to any allergic disease, a screening system for searching for a compound having an effect of restoring it will be developed. Can be built.
- the expression level increase refers to a state in which the target gene is introduced as a foreign gene and is forcibly expressed, or the transcription of an indicator gene originally provided in the test animal and the translation into a protein are enhanced. It means any of the following states: and the state in which the degradation of the protein as a translation product is suppressed.
- the expression level of the gene can be confirmed, for example, by quantitative PCR as described in Examples.
- the activity of the protein as a translation product can be confirmed by comparison with a normal state.
- a typical transgenic animal is an animal into which a target gene has been introduced and forcibly expressed.
- transgenic animals include, for example, animals in which a mutation is introduced into a gene coding region to enhance its activity or to be modified to an amino acid sequence that is hardly degraded.
- the mutation in the amino acid sequence can be substitution, deletion, insertion, or addition.
- the expression itself of the gene of the present invention can be regulated by mutating the transcriptional regulatory region of the gene.
- Methods for obtaining transgenic animals for specific genes are known. That is, a method in which a gene and an egg are mixed and treated with calcium phosphate, or a method in which a gene is directly introduced into a nucleus of a pronuclear stage egg with a micropipette under a phase contrast microscope (microinjection method, US Pat. No. 4,873,191).
- Transgenic animals can be obtained by methods such as using embryonic stem cells (ES cells).
- ES cells embryonic stem cells
- a method of introducing a gene into a retroviral vector and infecting an egg and a method of introducing a gene into an egg via sperm, such as a sperm vector method, have also been developed.
- the sperm vector method is a gene recombination method in which a foreign gene is introduced into sperm cells by a method such as attaching or electroporating sperm and then fertilizing the egg to introduce the foreign gene. M. Lavitranoet et al. Cell, 57, 717, 1989).
- the expression level of the exogenous Matk gene in the transgenic animal can be adjusted by administering the substance. Can be.
- transgenic animal used as the allergic disease model animal of the present invention can be prepared using any vertebrate other than human. Specifically, transgenic animals have been created in which vertebrates such as mice, rats, rabbits, egrets, miniature pigs, goats, sheep, and birds have been introduced or whose expression levels have been altered. .
- the present invention relates to a method for screening a candidate drug for treating an allergic disease.
- the expression level of the Matk gene is significantly increased in patients with allergic diseases. Therefore, a therapeutic agent for allergic diseases can be obtained by selecting a compound that can reduce the expression level of the gene.
- the compound that decreases the expression level of a gene is a compound that acts to inhibit any one of the steps of transcription, translation, and expression of protein activity.
- the method of screening for a candidate compound for treating allergic diseases according to the present invention comprises: It can be done either in vitro or in vitro.
- This screen “Jung” can be performed, for example, according to the following steps.
- a Matk gene or a gene functionally equivalent to the Matk gene can be used as the indicator gene.
- a functionally equivalent gene is a gene that encodes a protein having the same activity as that of the protein encoded by the indicator gene.
- a representative example of a functionally equivalent gene is the power gene of the indicator gene in the animal species that the test animal originally has.
- an allergic disease model animal can be used as a test animal in the screening method of the present invention.
- Allergic disease model animals are known.
- a spontaneous dermatitis model using NC / Nga mice has been reported.
- a total of eight doses of mite antigen (5 ⁇ g / ear) administered to the pinna of the mouse at 2-3 day intervals can induce symptoms very similar to human atopic dermatitis after 2 weeks.
- the screening according to the present invention can be carried out by administering a scavenger conjugate to this system and tracking changes in the expression level of the indicator gene of the present invention.
- the drug candidate compound is administered to the test animal, and the effect of the compound on the expression of the indicator gene in the biological sample derived from the test animal is monitored. Impact can be assessed. Fluctuations in the expression level of the indicator gene in a biological sample derived from a test animal can be monitored by the same method as the above-described test method of the present invention. Furthermore, based on the results of this evaluation, drug candidate compounds that reduce the expression level of the indicator gene are selected. Then, drug candidate compounds can be stared.
- the screening according to the present invention can be performed by collecting a biological sample from a test animal and comparing the expression level of the indicator gene with a control to which no scavenging compound is administered.
- raw materials use smooth muscle cells, keratinocytes, nasal mucosal epithelial cells, intestinal epithelial cells, mononuclear cells, lymphocytes, mast cells, eosinophils, basophils, or neutrophils. be able to. Methods for collecting and preparing these biological samples are known.
- Such a screen allows selection of drugs that participate in the expression of the indicator gene in various forms. Specifically, for example, drug candidate compounds having the following actions can be found.
- a method of contacting a candidate compound with a cell that expresses an indicator gene and selecting a compound that reduces the expression level of the gene can be mentioned.
- This screening can be performed, for example, according to the following steps.
- cells that express the indicator gene can be obtained by inserting the indicator gene into an appropriate expression vector and introducing the vector into an appropriate host cell.
- Usable vectors and host cells may be any as long as they can express the gene of the present invention.
- Escherichia coli, yeast, insect cells, animal cells, and the like can be exemplified as host cells in the host-one-vector ⁇ "system. be able to.
- Examples of a method for introducing a vector into a host include a biological method, a physical method, and a chemical method.
- Biological methods include, for example, a method using a viral vector, a method using a specific receptor, a cell fusion method (HVJ (Sendai virus), polyethylene glycol (PEG), an electric cell fusion method, and micronucleus fusion. Method (transfer of chromosomes)).
- Examples of the physical method include a microinjection method, an electroporation method, and a method using a gene particle gun.
- Chemical methods include the calcium phosphate precipitation method, liposome method, DEAE dextran method, protoplast method, erythrocyte ghost method, erythrocyte membrane ghost method, and microcapsule method. '
- peripheral blood leukocytes and leukocyte-derived strain cells can be used as cells expressing the indicator gene.
- leukocyte cells include mononuclear cells and immature neutrophils.
- Granulocyte cell lines are suitable for the screening method of the present invention.
- the granulocyte cell line HL- 60, KU812, AML14. 3D10 S K562, KG- 1, RPMI6666, or TF-1 like Ru can be used.
- a candidate compound is added to the established leukocyte cells. Thereafter, the expression level of the indicator gene in the established leukocyte cells is measured, and a compound that reduces the expression level of the gene is selected in comparison with a control in which the candidate compound is not contacted.
- the expression level of the indicator gene can be compared not only by the expression level of the protein encoded by the gene, but also by detecting the corresponding mRNA.
- the mRNA sample preparation step described above is performed instead of the protein sample preparation step. Detection of mRNA or protein can be performed by a known method as described above.
- a transcriptional regulatory region of the gene of the present invention can be obtained, and a reporter attestation system can be constructed.
- the reporter atsey system refers to an atsey system for screening for a transcriptional regulatory factor acting on the transcriptional regulatory region, using the expression level of a reporter gene located downstream of the transcriptional regulatory region as an index.
- the present invention relates to a method for screening a therapeutic agent for an allergic disease, comprising the following steps, wherein the indicator gene is Matk or a gene functionally equivalent to Matk.
- transcription control region examples include a promoter, an enhancer, and CMT pox and TATA bottus which are usually found in a promoter region.
- the transcription regulatory region of the Matk gene in the present invention can be obtained as follows. That is, first, based on the nucleotide sequence of the cDNA disclosed in the present invention, screening is performed from a human genomic DNA library such as a BAC library or a YAC library by a method using PCR or hybridization to perform cDNA screening. Obtain a genomic DNA clone containing the sequence. Based on the sequence of the obtained genomic DNA, the transcription control region of the cDNA disclosed in the present invention is estimated, and the transcription control region is obtained. Cloning the resulting transcriptional regulatory region so that it is located upstream of the reporter gene Build a reporter construct. The resulting reporter construct is introduced into a cultured cell line to obtain a transformant for screening. The screening according to the present invention can be performed by contacting the transformant with a candidate compound and selecting a compound that controls the expression of a reporter gene as compared to a control not contacting the candidate compound. .
- a screening method based on the activity of an indicator protein can also be used. That is, the present invention relates to a method for screening a therapeutic agent for an allergic disease, which comprises the following steps, wherein the indicator gene is Matk or a gene functionally equivalent to Matk.
- Matk which is the indicator protein in the present invention
- this activity as an index, compounds having an activity of inhibiting the activity can be screened. Compounds obtained in this way suppress the action of Matk. As a result, allergic diseases can be controlled through inhibition of the indicator protein whose expression is induced in mononuclear cells.
- test candidate substances used in these screenings include compound preparations synthesized by existing chemical methods such as steroid derivatives, compound preparations synthesized by combinatorial chemistry, animal and plant tissue extracts or microorganisms. Examples include a mixture containing a plurality of compounds such as a culture, and a sample purified from them.
- kits include substrate compounds used to detect the label, Media and containers, positive and negative standard samples, as well as instructions describing how to use the kit can be packaged.
- the compound selected by the screening method of the present invention is useful as a therapeutic drug for allergic diseases.
- antisense DNA that can suppress the expression of the Matk gene is also useful as a therapeutic drug for allergic diseases.
- an antibody that recognizes a peptide containing an amino acid sequence of the Matk protein is also useful as a therapeutic agent for allergic diseases.
- the Matk gene is a gene whose expression is increased in mononuclear cells of patients with allergic diseases. Therefore, a therapeutic effect on allergic disease can be expected by suppressing the expression of the gene or suppressing the function of the protein encoded by the gene.
- the therapeutic agent for an allergic disease of the present invention contains the compound selected by the screening method as an active ingredient, and is produced by mixing with a physiologically acceptable carrier, excipient, diluent, or the like. Can be.
- the therapeutic agent for allergic disease of the present invention can be administered orally or parenterally for the purpose of improving allergic symptoms.
- dosage forms such as granules, powders, tablets, capsules, solvents, emulsions, and suspensions can be selected.
- Injections include subcutaneous, 3 ⁇ 4lt, intramuscular and intraperitoneal injections.
- a therapeutic effect can be achieved by introducing a gene encoding the protein into a living body using a gene therapy technique.
- Techniques for treating a disease by introducing a gene encoding a protein that produces a therapeutic effect into a living body and expressing the gene are known.
- antisense DNA can be incorporated downstream of a suitable promoter and administered as an antisense RNA expression vector.
- this expression vector is introduced into mononuclear cells of patients with an allergic disease, the antisense of these genes is expressed, and the therapeutic effect of allergic drugs is achieved by reducing the expression level of the genes. be able to.
- Methods for introducing an expression vector into mononuclear cells are known in vivo or ex'ra.
- the dosage varies depending on the age, sex, weight and condition of the patient, therapeutic effect, administration method, processing time, or the type of active ingredient contained in the pharmaceutical composition, but is usually once per adult.
- FIG. 1 is a graph showing the results of measuring the expression level of the Matk gene in patients with allergic diseases and healthy subjects.
- the upper graph shows the measured value (copy / ng RNA) corrected for the actin gene of each subject.
- the lower graph shows the results of statistical analysis between each group.
- V indicates a healthy person
- R indicates a steroid response group
- P indicates a weak steroid response group.
- the numbers represent the numbers of the subjects.
- FIG. 2 is a graph showing the results of measuring the expression level of the Matk gene in the steroid response group, the weak steroid response group, and healthy subjects.
- the upper graph shows the measured value (copy / ng RNA) corrected for the GAPDH gene of each subject.
- the lower graph shows the results of statistical analysis between each group.
- V indicates a healthy subject
- R indicates a steroid response group
- P indicates a weak steroid response group.
- the numbers represent the numbers of the subjects.
- Example 1 Selection of candidate genes using a DNA chip
- the two groups were collectively referred to as the "patient group." Heparin blood was collected. Next, the blood was centrifuged by specific gravity according to the following method to collect a mononuclear cell fraction, which was then cultured. 40 mL of whole blood (using heparin anticoagulant, final concentration 50 unit / mL) was placed in a centrifuge tube.
- T cells were stimulated using PHA (phytohaemagglutinin, lectin) and PMA (phorbol myri state acetate, phorpol ester) known as T cell mitogens as follows.
- Mononuclear cells were suspended in 10 mL of culture solution (RPMI1640 / 10% FCS) (1.5 10 6 cells /), and PHA (phytohaemagglutinin, final concentration SjUg / mL, SIGMA) / PMA (phorbol myri state acetate, (Final concentration 10 ng / ml, SIGMA) was added, and the reaction was performed at 37 ° C. under 5% CO 2 for 24 hours for 3 hours.
- the whole thigh was extracted according to the following method in order to quantify the amount of Matk (megakaryocyte associated tyrosine kinase) mRNA.
- RNA extraction For total RNA extraction, use the RA extraction kit IS0GEN (Tubongene) and follow the instructions. I went. The cultured cells were dissolved in 3 mL of Isogen (4 M guanidium thiocyanate, 25 mM sodium cyanate, 0.5% Sarcosyl, 0.1 M j3-mercaptoethanol, pH 7.0). The suction operation was performed 20 to 30 times with a 2.5 mL syringe with a 20G caterane injection needle. CHC1 3 was added 0. 6 mL (1/5 volume of Isogen), it was allowed to mix for 15 seconds with a mixer and allowed to stand for 2-3 minutes at room temperature. 15 4 ° C, 000 were r P m, 15 min centrifugation.
- Isogen M guanidium thiocyanate, 25 mM sodium cyanate, 0.5% Sarcosyl, 0.1 M j3-mercaptoethanol, pH 7.0.
- the suction operation was performed 20 to 30 times with a 2.5 mL
- the supernatant was transferred to a new tube, added with 3 / iL of Ethachinmate (Nitsubon Gene) and 1.5 mL of isopropanol (1/2 volume of Isogen), mixed by inversion, and allowed to stand at room temperature for 10 minutes.
- the mixture was centrifuged at 15,000 rpm at 4 ° C for 15 minutes, 3 mL of 75% ethanol (equivalent to Isogen) was added to the precipitate, and the mixture was centrifuged at 15,000 rpm at 4 ° C for 5 minutes.
- the precipitate was air dried or vacuum dried for 2-3 minutes.
- T7- (dT) 24 (Amersham Pharmacia Biotech) as a primer
- the T7- (dT) 24 primer consists of a nucleotide sequence obtained by adding d (T) 24 to the nucleotide sequence of the T7 promoter as follows.
- DNA Ligase, DNA polymerase I, and UNase H were purified to synthesize double-stranded cDNA. After extracting the cDNA with phenol / chloroform, it was passed through Phase Lock Gels to precipitate ethanol and purify.
- biotin-labeled cRNA was synthesized using the BioArray High Yield RNA Transcription Labeling Kit. CRNA was purified using an RNeasy Spin column (QIAGEN) and fragmented by heat treatment.
- GeneCMp R HuGeneFL manufactured by Af f ymetr ix Inc.
- GeneChip R HuGeneFL is composed of a probe having the nucleotide sequence you from approximately 5600 different human cDNA or EST.
- Streptavidin Phycoerythrin was added for staining. After washing, an antibody mixture of normal goat IgG and biotinylated goat anti-streptavidin IgG antibody was cast on the array. Furthermore, Streptavidin was again used to increase the fluorescence intensity.
- Phycoerythrin was added and stained. After washing, it was set on a scanner and analyzed with DNA chip analysis software.
- Expression fluorescence sensitivity was measured using Suite, a DNA chip analysis software, and data analysis was performed. First, Absolute analysis was performed on all chips, and the gene expression level of each sample used was measured.
- the fold change value is 3 or more.
- a gene whose Absolute call is A (absent) or M (marginal) in a healthy person is the gene of the patient's expression judgment base (marginal)
- Table 1 shows the classification of the genes selected by Gene Chip Comparison Analysis. fold change from raw data measured value 3 times or more and 1/3 times or less. Response group Weak response group
- Matk gene was selected as a gene whose expression level was increased in the patient group.
- the Matk gene is a gene closely related to atopic dermatitis disease, whose expression level is increased in patients with atopic dermatitis.
- PBMC peripheral blood mononuclear cell
- Mononuclear cells peripheral blood mononuclear cells
- Culture and RNA extraction were performed according to the method described in Example 1 (1).
- the reverse transcription reaction operation and the method of quantitative PCR are as follows.
- RNA solution was prepared by centrifuge at 15,000 rpm for 15 minutes at, and add 125 L of 80% ethanol to the precipitate. I got it. The mixture was centrifuged at 15,000 rpm for 5 minutes at 4 ° ⁇ , and the precipitate was air-dried or vacuum-dried for 2-3 minutes. 10 L of RNase-free DW was added and the absorbance was measured to obtain an RNA solution.
- Thigh solution 1-5 ⁇ ⁇ 01igo (dT) 12 _ 18 primer (GIBCO BRL) 500 rig, the BSA lg added, was 12 said with sterile distilled water. The mixture was allowed to stand at 70 ° C for 10 minutes, and cooled with ice. 4 L of 5XFirst Strand Buffer (GIBCO BRL), 2 ⁇ of 1M DTT, 1 ⁇ of 10 mM dNTPs (N G, A, T, C) were added and mixed. After heating at 42 ° C for 2 minutes, 200 units of SuperScriptll (GIBC0 BRL) was added and reacted at 42 ° C for 50 minutes.
- GBC0 BRL SuperScriptll
- the mixture was treated at 70 ° C for 15 minutes to inactivate reverse transcriptase, 2 units of RNaseH (GIBCO BRL) were added, and the mixture was heated at 37 ° C for 20 minutes. Sterile distilled water was added to make a 10 ng / VL cDNA solution, which was used for quantitative PCR.
- the primer has the following nucleotide sequence.
- Primer F 5'-CGT GTT CTT CTG CM CCT CAT-3, (SEQ ID NO: 2)
- Primer R 5, -CTT TGG TCT CAC CAG CTT GGT-3, (SEQ ID NO: 3)
- a reaction cycle was performed: 95 ° C, 15 seconds ⁇ 60 ° C, 1 minute as one cycle, and 40 cycles were performed. Then, 3% agarose gel (Agarose-1000, GIBCO-BRL) using electrophoresis buffer 1XTAE (50XTAE in 1 liter, 242 g of Tris base, 57.ml glacial acetic acid, containing 50 mM EDTA, pH 8.0) Using 5 ⁇ g / mL ethidium mouth, electrophoresis was performed at a voltage of 100 V for 30 minutes. An 85 bp band of the PCR product was observed with a UV lamp.
- electrophoresis buffer 1XTAE 50XTAE in 1 liter, 242 g of Tris base, 57.ml glacial acetic acid, containing 50 mM EDTA, pH 8.0
- the PCR product was cut out from the gel using a QIAEX II Agarose Gel Extraction kit (QIAGEN) according to the instructions. After separating the PCR products on a 3% agarose gel, the target fragment was cut out with long-wavelength (316 nm) UV. Gel with a razor And transferred to a 1.5 ml tube (-250 mg gel). Six times the volume of BufferQXl (300 1 for 50 mg of ex. Gel) and 10 L of QIAEX II glass beads were added, and the mixture was stirred well using a vortex mixer for 30 seconds. The mixture was heated at 50 ° C. for 10 minutes. At this time, the mixture was mixed every few minutes, and it was confirmed that the mixture was yellow.
- QIAEX II Agarose Gel Extraction kit QIAEX II Agarose Gel Extraction kit
- the subclone plasmid DNA was prepared using Wizard Plus SV Minipreps DNA Purification System (Promega) according to the instructions. The white colonies were picked up, cultured at 37 ° C in 1 to 5 mL of 100 ⁇ g / mL ampicillin-LB medium, and centrifuged at 3,000 rpm for 6 minutes. The precipitate was suspended by adding 250 zL of the resuspended solution, and 250 Lysis solution was added thereto, followed by inverting 4 times. Add 10 ⁇ L of Alkaline Protease, 4 The mixture was mixed by inversion and allowed to stand at room temperature for 5 minutes.
- Gene expression quantification was performed using TaqMan Prop using the ABI PRISM 7700 System. Performed by real-time PCR and performed according to the instructions. The reaction was performed using TaqMan 1000 Reaction PCR Core reagents (Applied Biosystems) according to the instructions. Standard for creating a calibration curve samples gradient of at least 5 stages, 107 ⁇ ; created the L0 3 copies scratch. The number of n per sample was at least two.
- TaqMan probe 5,-(FAM) ACA TGG TGG AGC ATT ACA GCA AGG ACA A (TAMRA)
- TAMRA 6-carb oxy-te trame t hy lrhoaarai ne
- reaction cycle was performed as follows: 95 ° C, 15 seconds ⁇ 60 ° C, 1 minute, 1 cycle.
- a calibration curve is automatically created from the Ct (threshold cycles) value of the PCR amplification curve against the logarithmic value of the relative initial concentration of the standard sample, and the relative initial concentration of the unknown sample is calculated based on this. Calculated.
- Actin feed primer 5 '-TCA CCC ACA CTG TGC CCA TCT ACG A-3' (SEQ ID NO: 5)
- TaqMan probe 5 '-(FAM) ATGCCC-T (TAMRA) -CCCCCATGCCATCCTGCGTp-3' (SEQ ID NO: 7)
- GAPDH forward primer 5, -GAAGGTGAAGGTCGGAGT-3, (SEQ ID NO: 8)
- GAPDH reparse primer 5 GAAGATGGTGATGGGATTTC-3 '(SEQ ID NO: 9)
- GAPDH TaqMan probe 5,-(FAM) CMGCTTCCCGTTCTCAGCC (TAMRA) -3 '(Array number: 10)
- Fig. 1 The expression level (copy / ng RNA) of Matk gene captured by actin is shown in Fig. 1 (top), and the expression level of Matk gene (copy / ng RNA) corrected by GAPDH is shown in Fig. 2 (top). Table 2 shows the actual data.
- a Matk gene was found that showed a difference in expression levels between patients with allergic diseases and healthy subjects. By using the expression level of the gene as an index, a method for testing an allergic disease and a method for screening a compound for treating the disease have become possible.
- Matk gene of the present invention Since the expression of the Matk gene of the present invention is linked to the disease state, suppressing its expression is a target of therapeutic strategies for allergic 'I' living diseases, and for monitoring such new therapeutic methods. Is expected to be useful as a new clinical diagnostic index.
- the expression level of the Matk gene provided by the present invention can be easily determined regardless of the type of allergen. Therefore, the pathology of allergic reactions can be comprehensively grasped.
- the method of detecting allergy according to the present invention can analyze the expression level using a biological sample as a sample, and thus has low invasiveness to patients.
- highly sensitive measurement using a small amount of sample is possible.
- high throughput and low price are increasing year by year. Therefore, the method of testing for allergy according to the present invention is expected to be an important diagnostic method at the bedside in the near future. In this sense, the diagnostic value of this Matk gene is high.
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Abstract
L'invention concerne un gène de Matk qui révèle une différence de niveau d'expression entre des patients atteints d'une maladie allergique et des sujets sains, le gène de Makt présentant un niveau d'expression élevé chez les patients. On décrit donc une méthode qui permet d'analyser une maladie allergique en utilisant comme indication le niveau d'expression de ce gène dans des monocytes du sang périphérique. L'invention concerne en outre une méthode de criblage d'un composé destiné à traiter cette maladie.
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JPH11332567A (ja) * | 1998-05-22 | 1999-12-07 | Dai Ichi Seiyaku Co Ltd | アトピー体質の判定方法 |
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WO2003083139A1 (fr) * | 2002-04-03 | 2003-10-09 | Genox Research, Inc. | Procede de diagnostic de maladie allergique |
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