WO2008069342A1 - Probe for determination of allergenicity or anti-allergenicity - Google Patents

Probe for determination of allergenicity or anti-allergenicity

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Publication number
WO2008069342A1
WO2008069342A1 PCT/JP2007/073890 JP2007073890W WO2008069342A1 WO 2008069342 A1 WO2008069342 A1 WO 2008069342A1 JP 2007073890 W JP2007073890 W JP 2007073890W WO 2008069342 A1 WO2008069342 A1 WO 2008069342A1
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WO
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Patent type
Prior art keywords
gene
present
food
microarray
invention
Prior art date
Application number
PCT/JP2007/073890
Other languages
French (fr)
Japanese (ja)
Inventor
Masuko Kobori
Hiroshi Shinmoto
Tatsunobu Fukushima
Yuichiro Nagata
Original Assignee
Incorporated Administrative Agency National Agriculture And Food Research Organization
Mitsubishi Rayon Co., Ltd.
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52Assays involving cytokines

Abstract

Disclosed are: an oligonucleotide for the determination of allergenicity and/or anti-allergenicity, which comprises a nucleotide sequence capable of hybridizing with at least a part of the nucleotide sequence for a gene related to the allergic response to a food; a microarray for the determination of allergenicity and/or anti-allergenicity, which comprises the oligonucleotide arranged on a substrate; and a method for the determination of allergenicity using the microarray.

Description

Bright fine manual Arerugen or anti Arerugi one determining probe art

The present invention relates to oligonucleotide probes and microarray I in which they are arranged is used to determine the allergenic and or allergic food ingredients. BACKGROUND

In recent years, the introduction of crops of food without eating experience, such as an increase in the consumption of specific foods, with the change of diet life, the risk of food allergies is increasing. Currently, about 1 to 3 percent of the Japanese people, has been estimated to be a patient of food allergy (National Life Center). Also, consumers' needs for safety and reliability of food has been expanding year by year, particularly for containing allergen, ingredient labeling of processed food are required for a particular substance. In this way, the relationship between allergen and safety in food is at present, are becoming serious, the development of convenient and of good not allergenicity of the reproducibility of the determination method is a need for an unknown allergen.

Further, with the increase in allergic patients, the components contained in the food, it is required and has a high allergy suppressing or relaxing activity safe, scientifically elucidated Arerugi one inducing effect of food , it is also important to refer to eye the development of food that does not cause a Arerugi scratch.

Conventionally, as the allergenic determination method ELISA method has been used. However Na grounds, the method stops to detect known allergenic agent, In its unknown substances Nitsu can not detect the allergen. Therefore, in determining the allergenic food is quite have unknown crop that was has no alternative but to infer upon administration to prolonged animal convenient allergenic determination methods heretofore known have a les ^ also consider, when determining the allergenic Ya antiallergic effect of food components, combined effect of various foods, namely the effect of allergenicity when eaten in combination of a plurality of food is changed it is necessary, it is substantially difficult to do this by ELISA, to check such changes are considered to be possible only by analyzing the expression behavior of multiple genes.

In recent years, the progress of research on the genome of an organism information, including the human genome is markedly, especially in the field of medical care rather than base to take advantage of the actual diagnosis or treatment activities in application of this information, post-genome research is very actively row are we. In applications of this post-genome research and its, what is recognized as a very effective tool, a DNA microarray (also called a DNA chip). The DNA microarray is a tool pasted multiple gene fragments in a compartment with a fine small area, by detecting the presence or absence or the amount of gene fragments in a sample with the hybrida I See Chillon reaction between nucleic acids such as , it is possible to easily analyze the expression behavior of multiple genes.

DNA microarrays, present, those produced by a photolithography one method or spotting method has been used primarily as a research tool, "fiber-type DNA as applicants own platform which applies the fiber and resin technology microarray (trade name Jienoparu) "(reference 1-3) have been placed on the market is developed (URL: http: 〃 www.mr co.jp/genome/). The fiber-type DNA microarray, the adoption of own manufacturing process, a large amount production production can point characterized by a highly accurate DNA microarray low cost. Further, since the operation in the array of the manufacturing process does not require skill, there is no such variation in test results due to individual differences of the test personnel, it is not necessary to considering the individual difference of each experimental animal to further. Therefore, it is possible to easily obtain accurate data when using the fiber-type DNA microarray.

Meanwhile, as research on the development and inhibition of allergy, so far clarified food ingredients indicating the inflammatory cytokine production suppressing effect and degranulation inhibitory effect, etc. related to allergies inhibiting effect, the detailed mechanism of action review and clarification has been promoted (Ref. 4). As a result, the basic knowledge about the gene associated with the development and inhibition of allergic are obtained.

Recently, in the material has never been subjected to genetically modified foods and food, it is often unknown allergens with Ya antiallergic. However, these foods and the like are at present, it is more distributed in the market. Therefore, knowing the allergenic and anti-§-les-saving one of the food is very important to those of allergies. Whether they have these allergens, etc. is to chronically administered foods allergic unknown in experimental animals for more than several months, it is by connexion determined to observe allergic symptoms. However, even if studied to spend such time, the resulting result is has failed more than speculative. Reference document

1. Pat. No. 3,515,470

2. Pat. No. 3,510,882

3. Pat. No. 3,515,570

4. Kobori et al. Cell Death Differ., 11 (1) 123-130 (2004) DISCLOSURE OF THE INVENTION

The present invention aims at providing an allergen or provide and DNA microarrays in which they are arranged in a simple and highly reproducible genes determine antiallergic food ingredients. The present inventor has conducted extensive studies to solve the above problems, that combine the knowledge onset or inhibiting allergy by food ingredients, and DNA microarray technology, reproduces the allergen or anti-allergic food ingredients It found sexual well determined to that efficiently simple and highly reproducible genes. Further, it succeeded in developing them is arranged DNA microarrays, the present invention has been completed.

That is, the present invention in the nucleotide sequence of the genes associated with food allergy responses, comprising the nucleotide sequence capable of High Priestess soy at least a portion of the nucleotide sequence, in allergenic and Z or anti-allergic determination oligonucleotide Puropu is there. The genes associated with food allergy responses such as IL-l a gene, IL-1 13 gene, IL-2 gene, IL-18 gene, CCL2 gene, CCL3 gene, CCL4 gene, CCL 5 gene and TNF o! at least one selected from the group consisting of the genes can be mentioned. Further, in the present invention, allergenic and Roh or anti-allergic-size constant, for example those for type I allergies one or IV allergy.

Furthermore, the present invention, the oligonucleotide probe is placed foundation, a § allergens property and Z or anti Arerugi one determination microarray.

Furthermore, the present invention has a plurality of through holes, a them through holes in the microarray that have gel is maintained, the gel in the oligonucleotide professional one blanking is held, allergenicity and Z or anti Ru microarray der for allergic judgment.

In the microarray of the present invention, the oligonucleotide pro part is rather preferably is less than 1 0 0 0 type.

Furthermore, the present invention comprises the following steps, it is allergenic and / or anti-allergic determination method of the test substance:

(1) The test substance is administered to experimental animals or in contact, and Z or process a test substance is contacted with cells,

(2) extracting the biological substance from the experimental animals and Z or cells, and

(3) the biological substance or the preparation step of contacting the oligonucleotide professional one blanking or the microarray. Will be described embodiments of the present invention are described below. The following embodiments are merely illustrative for explain the present invention and are not intended to limit the invention to these embodiments alone. The present invention, without departing from the spirit thereof, are possible as in child in various forms.

Note that all of the publications cited herein, for example, prior art documents and publications, patent publications and other patent documents, entirely incorporated as a reference herein. The present specification encompasses the contents of Japanese Patent Application No. 2006-327852 specification made based on which the present application claims priority. The present invention, oligonucleotides pro part and a microphone port array capable of determining the presence or absence of allergy development or inhibition by food components efficiently and One effectively is provided. Furthermore, oligonucleotide probes and microarrays of the present invention, convenient for determining the allergenicity or anti-allergic effects in the unknown or determining or checking the allergenic food ingredients, development of functional food Ingredients it can also be used as a tool.

In the present invention, allergenic and Roh or pile allergic determination how food components, extracting nucleic acids from experimental animals or cultured cells were administered unknown foods allergic to a short period of about several days to several weeks and, wherein the contacting this heading group of genes and their microarray arranged these in the present invention. More methods of the present invention, a short time of evaluation of the allergic etc., labor saving, the results stabilization is achieved, in addition, it is possible to determine the combined effect of several foods. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram showing a group of genes expressed suppressed by TPCK.

Figure 2 is a graph showing the results of determining the state of allergy induced with LPS and OVA.

Figure 3 is a graph showing the results of determining the state of allergy induced with LPS and OVA.

Figure 4 is a graph showing the results of determining the state of allergy induced with LPS and PMA.

Figure 5 is a graph showing the results of determining the state of allergy induced with LPS and PMA. BEST MODE FOR CARRYING OUT THE INVENTION

The present invention, eating food candidates quality suspected of containing food or allergens including allergen, or one which is more complete that focusing on genes that varies upon contact with these foods, these genes or at about the microarray oligonucleotide probes are arranged having the nucleotide sequence Haiburidaizu portion thereof. That is, the present invention is a microarray O Rigo nucleotide professional one blanking associated with multiple food allergies response was arranged.

The "food allergy response", ingestion or nasal inhaled food component, or dietary components in contact with the skin causing cause excessive immune responses, various physical condition in individuals that ingested like food It refers to a disorder reaction to induce abnormal. Food allergies - The, (a) also cause symptoms of immediate by a specific I g E antibodies allergen, or (b) allergen-specific inflammatory T cells, delayed that by the eosinophils or macrophages there is one cause of swelling or organization Yabu壌. Food allergies, gastrointestinal tract, respiratory tract, easily develops skin, that in particular believed deeply associated with atopic skin 廇炎. Here, as the food containing the allergen, for example, eggs, milk, meat (beef, chicken, pork, etc.), grains (soy, rice, wheat, buckwheat, sesame, peanut, etc.), yam, fish and shellfish (Oh wabi, squid , salmon roe, shrimp, crab, salmon, mackerel, etc.), fruits (orange, Kiuifu roots, peaches, apples, figs, etc.) and the like.

As the allergen, for example, ovalbumin in egg, etc. Obomuko id, in the milk casein; etc. 6 lactoglobulin, grayed in wheat triazine, a like amylase inhibitors evening one, in peanuts for 2 S albumin etc., pulp albumin, tropomyosin, and the like in fish and shellfish. In the present invention, the term "oligonucleotide probe" refers to as a stylet for the detection of the gene expression level in connection with promoting or inhibiting the allergic response is changed. Such probes, cause immune response by ingestion of food with example allergens, originating when the allergens gene expression level is changed or inhibitor in order to suppress the allergic reaction, it is produced when produced oligonucleotides can be cited for the detection of the gene expression level is changed (hereinafter, referred Puropu).

It gene of interest detection is expressed amount in relation to the promotion or inhibition of allergic response is not particularly limited as long as the gene that change, a group of genes related to various cytokines, histamine, etc. It is preferred. Inter alia, genes useful minimum unit to determine the typical type I allergy and type IV allergy as food allele formic scratch.

Here, "type I allergy" is allergic reaction occurs after contact immediately with the antigen (allergen). Antibodies of IgE class, binds to Fe s receptions evening one on mast cells in the Fc part. When this bound antigen, Fc receptions evening one are bonded to each other through the antigen molecule. Thus, histamine from mast cells are released extracellularly, Roikotoryen, Chemical Medei et Isseki one such platelet activating factor is issued synthesis and release. Chemical media er Yuichi vascular hyperpermeability, smooth muscle contraction, lead and leukocyte run of expressed symptoms by resulting tissue response.

The "type IV allergy" refers to a tissue injury caused without antibody involvement by the reaction of antigen and T cells. T cells produce Rinhokai emissions such as interleukin (IL), thereby infiltration of macrophages Ya neutrophils, exudation of plasma by vascular hyperpermeability, results and fibroblast proliferation.

In the present invention, it directed to a group of genes related to such allergies. These genes, IL-1 (interleukin) alpha gene, IL-l jS gene, IL-2 gene, IL-4 gene, IL-8 gene, IL-10 gene, IL-18 gene, CCL2 ( chemokines) gene, CCL3 gene, CCL4 gene, CCL 5 gene, PTGS2 (prostaglandin - endoperoxide o sulfoxide synthase 2) and TNF (preferably tumor necrosis factor) shed genes, more preferably, IL-l o gene , IL-l j6 gene, IL-2 gene, EL-18 gene, COL2 gene, CC 3 gene, a gene shed CCL4 gene, CC 5 gene and TNF. These genes can be used in combination of the one or more. Preferably, it used to determine by observing the behavior of Te these genes to base.

Oligonucleotides used as probes in the present invention, among the nucleotide sequences of these genes are those as possible out to Haiburidizu at least part of the nucleotide sequence. That is, the probe of the present invention is one which is designed to be complementary to to a nucleotide sequence of the gene can be Haiburidizu when performing Haipuridaize one cane down to the gene in the test sample . Such probes, the c DNA of said gene was prepared, it may be arranged in a microarray. However, even if the type of proteins and peptides are different, some of the DNA encoding those proteins or peptides which may nucleotide sequence is present area you similar. Thus, Te is cowpea the base sequence of the probe selection, if the array is similar to each other occurs, can not be detected by distinguishing the plurality of genes in such nucleotide sequence. Therefore, the probe, it is preferable to design the nucleotide sequence of the region by selecting a region such that the nucleotide sequence specific to the target gene. It in the design of probes, in addition to selecting a specific region, the distance from the 3 'end is within 1 5 0 0 base, Tm (melting temperature) is at 6 0-8 0 , GC base content in the probe 4 0-6 0%, and it is preferable to design upon adding the formed and plastering of the secondary structure. Are shown in Table 1 you later for these conditions. To design the nucleotide sequence of the is between the gene of origin of the arrangement to the probe, the homology one nucleotide sequence between a search utilizing the existing programs, may be selected having low homology with location. Homology may be selected in designing the base sequence of the probe is less than 3 0%, preferably 25%, more preferably not more than 1 0%, where the length of the probe that is designed 1 0-2 0 0 base, is preferred properly 1 0-1 0 0 base. The probe can be using conventional oligonucleotide synthesis methods to prepare by chemical synthesis. Such probes are, for example, Probe Quest: as possible out to design the (R Dainakomu Inc.).

Probes are those which can detect the target gene and Haiburidizu when subjected to High Priestess die See Chillon the sample as described above. Therefore, when designing a pro portion, it is necessary to consider the stringency diene Sea in hybrida I See Chillon. By somewhat closer to stringent diene sea, even in the presence of base sequence regions similar between genes, it can be Hachii Buridizu distinguish between other different areas. Also, if the nucleotide sequence of the intergenic little different, can be gently set the stringency diene Sea.

The conditions for such stringent diene Sea, for example, in the case of tight condition is Haiburidize one Chillon under conditions of 6 5 ° C~6 8 ° C, if the mild conditions from 3 7 ° C 5 Haiburidize an Chillon under conditions of 5 ° C. In Haiburidize one Deployment condition in the present invention, the conditions of stringency, for example, Γ0.12Μ Tris -HCl O.12M NaCl / 0.05% Tween-20, 50 ° C ",

Γ0.12Μ Tris · HC1 / 0.12M NaCl / 0.05% Tween-20, 42 ° Cj, "0 · 12Μ Tris · HCl Z0.12M NaCl / 0.05% Tween-20, 37 ° C", more stringent conditions such as is, for example, "0.12M Tris · ΗΟΐΖ0.12Μ Ν & /0.05% Tween-20, 65 ° C", "0 · 12Μ Tris · HC1 / 0.12M NaCl / 0.05% Tween-20, 68 ° C", "0.06M Tris · HC1 / 0.06M NaCl / 0.05% Tween-20, 65 ° C "conditions, and the like.

More specifically, the addition of probes kept 65 ° C (149 ° F) over 1 hour to Haipuriddo formed, then, in 0.12M Tris' HCL / 0.12M NaCl / 05% Tween-20, washing 20 minutes at 65 ° C four times, finally, there is a method of performing one of 10 minutes washed with 0.12M Tris-HCl / 0.12M NaCl, 65 ° C. Hybrida I See Chillon, or the temperature at the time of cleaning by the above gel, it is possible to set a more stringent conditions. Those skilled in the art, such as salt concentration of the buffer, in addition to conditions such as temperature, other probe concentration, probe length, taking into account various conditions such as reaction time, and the child set conditions it can.

For detailed instructions Haiburidize one Chillon method, "Molecular Cloning, A Laboratory Manual 2nd ed.J (Cold Spring Harbor Press (1989,, the" Current Protocols in Molecular BiologyJ (John Wiley & Sons (1987-1997)) or the like in reference to can Rukoto. present invention, the base sequence of the detection target gene need not be a the nucleotide sequence of each gene as described above, the deletion part of the nucleotide sequence, substitution, mutation by insertion or the like occurs or it may be a. Therefore, the detection target gene includes a sequence complementary to a nucleotide sequence represented by SEQ ID NO described in table, and Haiburidizu under stringent conditions, and the activity of each gene (activity as inter one interleukin 1 activity such as CCL1) mutant gene having also can target, probe, bases such mutant gene Column as possible out also be designed as a basis. "Stringent conditions", "probe" can be Ru. Above applying the same conditions, is arranged. Probes plurality placed foundation foundation as generally referred to as DNA chip or DNA microarray. foundation, glass plate, a resin plate, a method of. arrangement silicon plate and the like is, the base plate surface by previously chemically modified bases how to expose a functional group, a method (spotting method) placing the probe by spot potting for the functional groups, or to synthesize successive probes silicon base shape by the light-induced solid phase reaction (photolithography graphic of the Act) It has been known.

Also has a plurality of through holes, microarray gel them through hole is held (hereinafter, referred to as through-hole array.) It is also preferably used. A through-hole array can be prepared, for example, by the following method (see JP 2 0 0 0 7 8 9 9 8 No.).

(1) drilling as parallel holes with a laser or the like made of resin proc is formed.

(2) introducing a gel precursor solution comprising a probe into the hole, the gelation reaction in the hole real Hodokosuru.

(3) repeated cutting a resin proc in a direction transverse cutting a hole.

Preferred examples of the through-hole array, the hollow fiber type microarray can be exemplified (Japanese Patent 2000-245461, JP 2001-239594, JP-see JP 2003-344400). For hollow fiber type microarray which can realize low cost microarray with high accuracy, it is preferable to use hollow fiber type microarray in the present invention. Hollow fiber type microarray can be produced more sequentially performing for example the following steps.

(1) to align the fiber by passing the pores of the plurality of jigs with holes sequence the same pattern as the desired hollow fiber.

(2) after it spreads to the width of the object distance jig to secure the fiber bundle and fill the matrix resin between the fibers. Thus, Proc fiber bundles are fixed by the resin (array) can be obtained.

(3) to the hollow portion of each fiber, by introducing a gel precursor solution comprising probes, gelation reaction is carried out in a hollow portion, to fix the probe to the gel.

(4) cutting in a direction intersecting the longitudinal direction of the fiber.

The cutting means that slicing in a direction crossing the block to the longitudinal direction, which makes it possible to obtain a thin piece of array. Probe is fixed to the fiber portion of the lamina. Method for manufacturing the fiber type microarray is described in JP-A-2001-239594, it is possible to produce a fiber-type microarray by reference to this publication. Also in the hollow portion is held gels such as acrylamide, Origonukure Ochido (probe) preferably are fixed (held) in the gel. The fixed Do you Yo thereof, probes are three-dimensionally arranged in the gel, allows you to maintain the 3-dimensional structure. Therefore, the surface than the probe in a plane microarray was bound on a glass slide coated with, detection efficiency is increased, it is possible to inspect the highly sensitive and reproducible.

Furthermore the number of types of probes are arranged in microarrays, one micro-on array (medium) 1 0 0 0 or fewer, preferably 5 0 0 or fewer, still more preferably 2 5 0 or fewer. More limiting thus arranged number gene, it can be detected with higher sensitivity aforementioned gene found in the present invention. In the present specification, the type of probes are distinguished by sequencing. Therefore the nucleotide sequence even probes of the same gene derived is identified as a different type for different even one. Next a method for inspecting the allergenic and Z or anti-allergic test substance.

The present invention comprises the following steps, it is allergenic and z or antiallergic inspection method of the test substance.

(1) administration of test substance or in contact with the experimental animals, and step of Se' the Roh or test substance to cells,

(2) extracting the biological substance from the experimental animals and Z or cells, and

(3) the biological substance or the preparation step of contacting the oligonucleotide or microarrays described above. The term "allergenicity", to function as an allergen in feeding time, refers to the nature of the foods that cause the results as allergic response. An "allergen" refers to a causative agent that causes an allergic reaction. Allergens are presented and processed by macrophages antigen recognition T cells, induces antigen-specific T cell and antibody production. Incidentally, the cell or type of antibody is inductive, and the symptoms of allergic reaction, the living body conditions are sensitized (e.g., a gene factors, nutrition) varies by or invasion route of allergen.

An "anti-allergic" means property of inhibiting allergic response. Because it contains a number of step in response of the § Rerugi one, it may be possible to us have suppressed allergy to each step. Therefore, the quality ones having antiallergic properties, as long as suppressing allergic response may function in any step of the development of allergy. The food containing a component having an anti-allergic, for example, tea, and the like, as the components having antiallergic one property, such as green tea catechins can be mentioned up. In the first step, by administration or contact with the test substance to an experimental animal, and or brought into contact with the cells. The "test substance" is a substance that is subject to inspection. For example foods, a solution which was homogenized, the crude purified product, and the like. Forms of administering or contacting the test substance (the test food) in laboratory animals is not intended to be limited to oral administration 及 beauty parenteral administration (injection, transdermal, etc.) may be either. Further, embodiments of contacting the cell, for example adding the candidate substance to the cell culture medium is intended to mean any to cultured cells with a candidate agent. The period of administering a test substance to an experimental animal may be a short-term administration of several days. When using cells is not limited to the phases of the cell cycle, it may be used any phase of the cell.

When administered parenterally the test substance (in particular when the injection), the test substance is a liquid, and also the case of a test substance into contact with cells, usually performs liquid culture. Therefore, homogenate the liquid food such as beverage when the test substance can be used by diluting with a left or a suitable buffer liquid, when solid and test substance in a suitable buffer or culture medium Ichitoshi can be diluted to a concentration suitable for subsequent culture or administration. The types of cells, for example, B cells, T cells, basophils, macrophages, allergy-related cells, etc., such as mast cells. When analyzing an anti-allergic, first, Ku Wakashi administering a candidate substance capable of eliciting a state of allergy like to experimental animals is contacted, and is contacted with Z or cells. This step, for example IL-1 Q! Gene, IL-l iS gene, IL-2 gene, IL-18 gene, the expression of all or part of the gene of CCL2 gene and TNF a gene enhances but it followed by administration or contacting a test substance with allergenic one inhibitory effect in experimental animals, and / or by contacting a cell, whether enhanced in all or part of the gene is suppressed the analyzes.

In the second step, extracting the biological substance from the experimental animals and Z or cell. The biological substance, which is DNA, RNA, proteins, peptides, peptide nucleic acid, lipid, antibody, or the like. These extraction methods can be suitably selected by those skilled in the art.

In a third step, the extracted biological substance or the preparation is contacted with the probes described above or microarray. The method of the present invention detects a signal emitted from the probe, or the preparation after the contact, comprising the step of determining allergenic and / or anti-allergic test substance based on a detection result of the signal. By "preparation", for example, those extracted biological substance labeled RI, a fluorescent or the like, nucleic acids were amplified using PCR, T7 amplification method is a fragmented nucleic acid, and the like. The biological substance or preparation by contacting the gene or microarray, it is possible to observe reactions with the probe.

The reaction conditions are the type of biological substance to be used is appropriately selected depending on the type, etc. of the probe. After completion of the reaction, by suitable detection means, to evaluate the reactivity of the bio-related substance and the probe. For example, if the biological substance is a fluorescent label, performs Arerugen and / or anti Arerugi one of evaluation by measuring the fluorescence intensity Ri by the fluorescence microscope. Here, the "evaluation", for example to quantify the expression level from the measured fluorescence intensity, certain types of gene expressing upon exposure to the test substance by comparison with control group, and the expression level it is to identify.

Probes of the present invention can be used to allergenic and / or anti Arerugi one determination against type I allergy or IV allergy. By using probes or microarrays of the present invention, it becomes possible to make determination and Ken查 convenient allergen properties, it is easy to the proper display on the food with allergenicity. This is the building of a healthy diet, allergy prevention, help to build the confidence to food companies, leading to the development of the industry.

Further, it becomes possible to easily determine and check antiallergic the present invention can be used in the screening of candidate substances pharmaceuticals, food research site. Accordingly, the present invention, novel pharmaceuticals, streamlining or faster novel functional food development, reduction of development risk, in that it can be reduced development costs, probes, microarrays and methods of the present invention is very useful .

Furthermore, the present invention comprising said probes or microarray, to provide allergenic 及 Binomatawa antiallergic determination kit Bok.

Kits of the present invention, in addition to the microarray may contain other components. Examples of other components, for example, fluorescent reagents, restriction enzymes, various buffer solutions, sterile water, detergents, surfactants, and experimental manipulation manual (manual) or the like.

Hereinafter, a more detailed description of the present invention through examples. However, the present invention is not limited by these examples.

Example 1

Production of microarrays

1. Preparation of the arrangement probe

First, use to select through-hole type of the microarray as microarrays used in the experiment, as the probe nucleic acid molecules for placement in DNA microarrays, having the sequence information set forth in Table 1 5 'terminus vinyl nucleic acid molecule (65mer) It had.

Table 1. In No.104 is a probe corresponding to the IL-1 shed gene, No.105 is IL- 1 ^ 6 gene, No.107 is IL · 2 gene, No. L03 is IL- 18 gene, No .19 is CCL2 gene, No.24 is CCL3 gene, No.25 is CCL4 gene, No.26 is the CCL 5 genetic Kooyobi No.208 is a probe corresponding to the TNF gene.

table 1

68C.0 / .00Zdf / X3d monument £ 690 / 800Z OAV OS

68C.0 / .00Zdf / 13d monument C690 / 800Z ΟΛ \ C.0 / .00Jdf / X3d monument £ 690 / 800Z OAV Example 2

1. Test sample preparation

In the present embodiment, using LPS (Lipopolysaccharide) as a substance capable of eliciting a state of allergy-like, TPCK as a substance with inhibiting effect on allergic-like state (Modified: 1J- (tosylamido - 2 - phenyl) ethyl chloro methyl with ketone, as a cell: RAW246.7 cells (mouse macrophage-derived cultured cells) had use of.

RAW246.7 cells LPS stimulation alone, and LPS-stimulated and incubated for 6 hours under TPCKlOOpmol presence, respectively the cells were harvested and respectively extracted Total RNA. Extraction of Total RNA was performed using the RNeasy MinElute Kit (QIAGEN Inc.). Alone stimulation group of LPS stimulation Arerugi one induced model group, groups cultured under TPCK coexistence was Areru Guy inhibition model group.

Using Total RNA of each group, the Biotin labeled aRNA for microarray hybridizing Zeshiyon of the present invention were prepared and subjected to Haiburidize one Chillon. Preparation of Biotin-labeled aRNA In its use the Message Amp II Biotin Kit (Ambion, Inc.) according to a predetermined method.

2. microarray According Atsusi

The High Priestess die See Chillon buffer solution was prepared as follows.

(0.12M TNT solution: 1000ml)

1M Tris-HCl 120ml

1M NaCl 120ml

0.5% Tween20 100ml

Female up to 1000m 1 with distilled water

The Biotin-labeled aRNA, 15 are suspended in g Z LSO 1 to hybridization one Chillon buffer first solution (0.12 M TNT solution), rows 1 7 hour hybridization reaction at 65 using Mitsubishi Rayon Co., Ltd. microarrays (GenoPa) One was. After the reaction chip performs washed twice for 2 0 min at 65 ° C using wash buffer one solution (0.12 M TNT solution), and finally washed for 10 minutes in 2 x SSC. Detection of the chip was performed using a Mitsubishi Reiyon manufactured by the detector.

Fluorescent label, Cy5-Streptavdin that the nitrous et beforehand distilled water was dissolved in lmg Z ml of (GE Healthcare Corp.) were added 5ml diluted with distilled water to 1/500, it was carried out by immersing the microarray for 30 minutes . Microarray after immersion for 5 minutes by 0.12 M TNT solution 5 ml, it was subjected to four washes, by Mitsubishi Rayon Co. DNA chip detector, and fluorescence intensity was measured.

3. DNA chip by the analysis results

The measured fluorescence intensity, performs a fluorescence intensity correction referenced to each group GAPD gene (NM_008084), and comparing the fluorescence intensity of the target gene in each group.

As a result, a the GAPD gene (fluorescent intensity value of the fluorescence intensity values ​​/ TPCK stimulation group of LPS alone stimulated group) (1 4 7 1 5 7/1 4 5 9 5 3), the ratio was 1.01 . If 0, expressed in their order relative expression differences GAPD log (base 2), the results of other genes was determined relative expression differences (Figure 1).

From FIG. 1, BLNK a gene group that has been enhanced by LPS stimulation, Camk2d, Caspll, CCL2, CCL5, F52, Gbpl, IL- lOra, IL- 18, IL-1, IL- 1 β, Lyn, Trafl, Trim30 is it was shown to be inhibited by TPCK stimulation.

Example 3

In the present embodiment, allergic-like state as a substance capable of eliciting an LPS (Lipopolysaccharide: Gram negative bacteria lipopolysaccharides) and OVA (egg white albumin: ovalbumin) were used. Incidentally, LPS is capable of inducing a characteristic response to elicit an inflammatory response, so to speak IV Kata Rerugi scratch. Also the OVA become allergen food allergy in the oral child may induce characteristic response to type I allergy.

Them material was administered to mice under the following conditions, spleens were harvested, mRNA of spleen cells was measured by microarray. The results are shown in FIGS.

<Experimental conditions> • Use mouse C 57 BL early 8-week-old

[LPS]

'Oral conditions: 1 dose, 0 days only, oral administration of l OmgZml to 0. 2ml mouse.

Spleens removed after 4 hours.

COVA)

'Oral conditions: 2 doses, day 0, day 8, oral administration of 10mg / ml in 0. 2ml mouse.

• removal of the spleen after 21 days.

[RNA extraction and labeling]

• was treated in the same manner as in Example 2.

'Microarray According Atsusi and DNA chip by analysis results was performed in the same manner as in Example 2.

As a result, to express significantly in LPS stimulated IL-la.IL-l | was found that S, CCL2, CCL3, CCL4, CCL5, TNFo a!. Also at the time of OVA administered IL-2, IL-18 has been found to TakashiSusumu (Figure 2).

That is, in the case of type IV allergy (IL-lQ!, IL-li6, TNFQi, CCL2, etc.) is increased, in the case of type I allergy is suggested that IL-2, IL-18 is enhanced, these by observing the expression gene, possible to determine the type of food allergy and Do ivy.

Example 4

In the present embodiment, LPS as a substance capable of eliciting a state of allergy-like (Lipopolysaccharide: g-negative bacteria-derived Li Po polysaccharides) and with PMA (12-O-Tetradecanoylp orbol 13-acetate). Incidentally, LPS can induce to cause attract the inflammatory response, so to speak] characteristic response to V-type allergy. The PMA promotes various sites force in secretion induces different reaction type IV allergy. Those substances were stimulated by addition to the mouse cells under the following conditions. Extracts Total UNA from these cell groups, the mRNA expression of each gene was measured by microarray. The results are shown in FIGS.

'Use cell RAW 2 6 4. 7 cells (mouse macrophage-like cells)

Ingredients experimental conditions>

[LPS]

• by adding LPS to 1 X 1 0 6 cells / ml of cells at a final concentration of 1 0 g / m 1 stimulation, RNA is extracted by collecting the cells after 6 hours

CPMA]

· 1 X 1 0 6 cells / ml of cells added to stimulate the PMA at a final concentration of 1 g / m 1, extracted RNA by harvesting cells after 6 hours

[RNA extraction and labeling]

• was treated in the same manner as in Example 2. . I went to like.

As a result, in LPS-stimulated genes in Figure 4 is not significantly expressed in are so elevated IL-l Q !, IL-2 ,, CCL2 in force PMA stimulation enhanced is permitted.

The results show that IL-la, observing the IL-2, CCL2 is considered to be effective to the type determination of allergy. Industrial Applicability

The present invention, oligonucleotide probes and microarrays capable of determining the presence or absence of allergy development or inhibition by food components efficiently and One effectively is provided. Furthermore, oligonucleotide probes and microarrays of the present invention, convenient for determining the allergenicity or anti-allergic effects in the unknown or determining or checking the allergenic food ingredients, development of functional food Ingredients it can also be used as a tool. Sequence Listing Free Text SEQ ID NO: 1 to 2 2 1: synthetic DNA

Claims

The scope of the claims
1. In the nucleotide sequence of the genes associated with food allergy responses, comprising the nucleotide sequence capable of High Priestess soy at least a portion of the nucleotide sequence, Arerugen property and Z or anti Arerugi one determination for oligonucleotide probes.
2. Genes associated with food allergy response, IL-l o! Gene, IL-l iS gene, IL-2 gene,] X-18 gene, CCL2 gene, CC 3 gene, CCL4 gene, CCL 5 gene and TNF is at least one selected from the group consisting of a gene, 請 Motomeko 1 wherein the oligonucleotide pro part.
3. Allergenic and / or anti-allergic determination, I-type allergy or IV Kata Rerugi is for one of claims 1 or 2, wherein the oligonucleotide probe.
4. Oligonucleotide probes according to any one of claims 1 to 3 is arranged in the base, Arerugen property and Z or anti Arerugi one determination microarray.
5. A plurality of through holes, a microarray gel they through hole is held, an oligonucleotide probe according to any one of claims 1 to 3 wherein the gel is held, allergenicity and Z or anti-allergic determination microarray.
6. Oligonucleotide probe is less than 1 0 0 0 type, the microarray according to claim 4 or 5.
. 7, comprising the steps of a test substance Arerugen and / or anti Arerugi one of determine measuring method:
(1) Step a test substance is administered to experimental animals or in contact, and or the test substance is contacted with cells,
(2) extracting the biological substance from the experimental animals and / or cells, as well as
(3) the biological substance or the preparation step of contacting the microphone port array according to any one of the oligonucleotide probes or claim 4-6 according to any one of claims 1 to 3.
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