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

Probe for determination of allergenicity or anti-allergenicity Download PDF

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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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Prior art keywords
gene
microarray
allergenicity
probe
allergy
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PCT/JP2007/073890
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French (fr)
Japanese (ja)
Inventor
Masuko Kobori
Hiroshi Shinmoto
Tatsunobu Fukushima
Yuichiro Nagata
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Incorporated Administrative Agency National Agriculture And Food Research Organization
Mitsubishi Rayon Co., Ltd.
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Priority to JP2007558378A priority Critical patent/JP5965569B2/en
Publication of WO2008069342A1 publication Critical patent/WO2008069342A1/en

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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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines

Definitions

  • the present invention relates to oligonucleotide probes used for determining the allergenicity and / or allergenicity of food ingredients, and microarrays on which they are arranged.
  • ingredients in foods are required to have highly safe allergy suppression or alleviation action, and scientifically elucidated the allergic action of foods. It is also important to aim to develop foods that do not cause allergies.
  • ELISA has been used as a method for determining allergenicity.
  • this method only detects known allergenic substances and cannot detect allergens for unknown substances. For this reason, there is no method to determine the allergenicity of an unknown crop that has never been eaten, other than to estimate it by long-term administration to animals. Not known les ⁇
  • allergenic anti-allergic effects of food ingredients It is necessary to consider the combined effects of foods, that is, the effect that allergenicity changes when multiple foods are used in combination, but this is practically difficult to perform by ELISA, and such changes are This can only be confirmed by analyzing the expression behavior of multiple genes.
  • a DNA microarray (also called a DNA chip) is recognized as an extremely effective tool in this post-genome research and its application fields.
  • a DNA microarray is a tool in which a large number of gene fragments are attached to a section having a small area. By detecting the presence or amount of gene fragments in a sample by a hybridization reaction between nucleic acids. It is possible to easily analyze the expression behavior of multiple genes.
  • DNA microarrays manufactured by a single photolithography method or spotting method are mainly used as a research tool.
  • Microarray (trade name Dienopearl) "(references 1-3) has been developed and marketed (URL: http: ⁇ www.mr co.jp/genome/).
  • This fiber-type DNA microarray is characterized by the ability to mass-produce high-precision DNA microarrays at low cost by adopting an original manufacturing method.
  • the operation in the array manufacturing process does not require skill, there is no variation in test results due to individual differences among test personnel, and there is no need to take into account individual differences among laboratory animals. Therefore, accurate data can be easily obtained by using the fiber DNA microarray.
  • An object of the present invention is to provide a simple and highly reproducible gene group for determining the allergenicity or antiallergenicity of food components and a DNA microarray in which they are arranged.
  • the present inventor has reproduced allergens or antiallergenicity of food ingredients by combining knowledge about the onset or suppression of allergies caused by food ingredients with DNA microarray technology.
  • An efficient, simple and highly reproducible gene group was determined.
  • the present inventors have succeeded in developing a DNA microarray in which they are arranged, thereby completing the present invention.
  • the present invention is an oligonucleotide probe for determining allergenicity and Z or antiallergenicity, comprising a nucleotide sequence that can be hybridized to at least a part of the nucleotide sequence of a gene related to a food allergy response.
  • genes related to food allergy response include IL-la gene, IL-1 13 gene, IL-2 gene, IL-18 gene, CCL2 gene, CCL3 gene, CCL4 gene, CCL 5 gene and TNF o! And at least one selected from the group consisting of genes.
  • the allergenicity and anti-allergic determination is for, for example, type I allergy or type IV allergy.
  • the present invention is a microarray for determining allergenicity and Z or antiallergenicity, wherein the oligonucleotide probe is arranged on a base.
  • the present invention provides a microarray having a plurality of through-holes and a gel held in the through-holes, wherein the oligonucleotide probe is held in the gel. It is a microarray for determining allergenicity.
  • the number of oligonucleotide probes is preferably 100 or less.
  • the present invention is a method for determining the allergenicity and / or antiallergenicity of a test substance, comprising the following steps:
  • an oligonucleotide probe and a microphone mouth array capable of efficiently and effectively determining the presence or absence of an allergy onset or an inhibitory action due to food components.
  • the oligonucleotide probe and microarray of the present invention are easy to determine or test the allergenicity of unknown food ingredients, or to determine allergenic or antiallergic effects in the development stage of functional food ingredients. It can also be used as a tool.
  • the method for determining the allergenicity of food ingredients and the allergenicity of piles or piles is based on the extraction of nucleic acids from experimental animals or cultured cells administered with unknown allergenic foods in a short period of time from several days to several weeks This is characterized in that it is brought into contact with a gene group found in the present invention or a microarray on which these genes are arranged. According to the method of the present invention, it is possible to shorten the evaluation of allergy and the like, save labor, and stabilize the results. Further, the combined effect of several kinds of foods can be determined.
  • FIG. 1 is a diagram showing a group of genes whose expression was suppressed by TPCK.
  • FIG. 2 is a diagram showing the results of determining the state of allergenicity using LPS and OVA.
  • FIG. 3 is a diagram showing the results of determining the state of allergenicity using LPS and OVA.
  • FIG. 4 is a diagram showing the results of determining the state of allergenicity using LPS and PMA.
  • FIG. 5 is a diagram showing the results of determining the state of allergenicity using LPS and PMA. BEST MODE FOR CARRYING OUT THE INVENTION
  • the present invention has been completed by paying attention to genes that change when eating food products containing allergens or food candidate substances that are thought to contain allergens, or coming into contact with these foods.
  • the present invention relates to a microarray in which oligonucleotide probes having a base sequence that hybridizes to a part thereof are arranged. That is, the present invention is a microarray in which oligonucleotide probes related to a plurality of food allergic responses are arranged.
  • Food allergic response refers to food components that are ingested or nasally inhaled, or that cause excessive immune responses due to food components that come into contact with the skin, and have various physical conditions in individuals who have consumed food. It refers to a disordered reaction that induces abnormalities. 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 May cause swelling or tissue destruction. Food allergies tend to occur in the gastrointestinal tract, respiratory tract, and skin, and are thought to be closely related to atopic dermatitis.
  • examples of foods containing allergens include eggs, milk, meat (beef, chicken, pork, etc.), cereals (soybean, rice, wheat, buckwheat, sesame, peanuts, etc.), sweet potatoes, seafood (abalone, squid) , How much, shrimp, crab, salmon, mackerel, etc.), fruits (orange, kiwi roots, peaches, apples, figs, etc.).
  • oligonucleotide probe refers to a probe that serves as a probe for detecting a gene whose expression level changes in association with promotion or suppression of an allergic response.
  • a probe for example, when an allergic substance is produced by ingesting an allergen-containing food, an expression level changes, or when an inhibitory substance is produced to suppress an allergic reaction.
  • Examples include oligonucleotides for detecting genes whose actual amount changes (hereinafter, Called a probe).
  • the gene to be detected is not particularly limited as long as it is a gene whose expression level changes in relation to the promotion or suppression of allergic response, but it is a gene group related to various cytokines, histamine, etc. Is preferred. In particular, it is a gene group of the smallest unit useful for discriminating type I allergy and type IV allergy, which is a typical food allergy.
  • type I allergy is an allergy that reacts immediately after contact with an antigen (allergen).
  • IgE class antibodies bind to Fe s receptors on mast cells at their Fc part.
  • the Fc receptors bind through the antigen molecule.
  • histamine is released from mast cells to the outside of the cell, and chemical media such as leukotriene and platelet activating factor are synthesized and released.
  • Chemical mediator Yui leads to increased vascular permeability, smooth muscle contraction, leukocyte chemotaxis, and as a result, symptoms appear due to tissue reaction.
  • Type IV allergy refers to tissue injury caused by the reaction between an antigen and T cells without antibody involvement.
  • T cells produce lymphokines such as interleukin (IL), which leads to macrophage infiltration of neutrophils, plasma exudation due to increased vascular permeability, and proliferation of fibroblasts.
  • IL interleukin
  • the gene group related to such allergy is the subject.
  • These genes include IL-1 (interleukin) ⁇ gene, IL-l jS gene, IL-2 gene, IL-4 gene, IL-8 gene, IL-10 gene, IL-18 gene, CCL2 ( Chemokines) genes, CCL3 genes, CCL4 genes, CCL 5 genes, PTGS2 (prostaglandin-endoperoxide synthase 2) and TNF (tumor necrosis factor) genes are preferred, more preferably IL-lo! Genes IL-6 gene, IL-2 gene, EL-18 gene, COL2 gene, CC3 gene, CCL4 gene, CC5 gene and TNF gene. These genes can be used alone or in combination. Preferably, the behavior of all these genes is observed and used for judgment.
  • the oligonucleotide used as a probe in the present invention has these genetics.
  • the probe of the present invention means a probe designed so as to be complementary to the base sequence of the gene and capable of hybridizing when performing hybridization with the gene in the test sample.
  • cDNA of the above gene may be prepared and placed in a microarray as it is.
  • the types of proteins and peptides are different, there may be a region with a similar base sequence in the DNA encoding the protein or peptide.
  • the sequences may be similar to each other, and such a base sequence cannot distinguish and detect a plurality of genes. Therefore, it is preferable that the probe selects a region that has a base sequence specific to the target gene and designs the base sequence of that region.
  • the distance from the 3 terminal must be within 1 500 bases and the Tm (melting temperature) must be 60 to 80 It is preferable to design in consideration of the GC base content in the probe of 40 to 60% and the ease of secondary structure formation. These conditions are shown in Table 1 below.
  • the homology that can be selected when designing the base sequence of the probe is 30% or less, preferably 25% or less, more preferably 10% or less.
  • the length of the designed probe is 1 It is 0 to 200 bases, preferably 10 to 100 bases.
  • the probe can be prepared by chemical synthesis using a normal oligonucleotide synthesis method. Such a probe can be designed by, for example, Probe Quest (registered trademark: manufactured by Dynacom).
  • stringency conditions include, for example, hybridization under conditions of 65 ° C to 68 ° C under tight conditions, and 37 ° C to 5 ° under mild conditions. Hybridization under conditions of 5 ° C.
  • stringent conditions include, for example, ⁇ 0.12 ⁇ Tris-HCl O.12M NaCl / 0.05% Tween-20, 50 ° C. ”
  • the probe is added and kept at 65 ° C for 1 hour or longer to form a hybrid, and then washed in 0.12M Tris'HCL / 0.12M NaCl / 05% Tween-20 at 65 ° C for 20 minutes. There is also a method of washing once for 10 minutes at 65 ° C in 0.12M Tris-HCl / 0.12M NaCl. More stringent conditions can be set by raising the temperature during hybridization or washing. A person skilled in the art can set conditions in consideration of other conditions such as probe concentration, probe length, and reaction time in addition to conditions such as salt concentration and temperature of the buffer. it can.
  • the base sequence of the target gene for detection need not be the base sequence itself of each gene described above, and may be a part of the base sequence that has been mutated by deletion, substitution, insertion, or the like. .
  • the target gene for detection hybridizes with a sequence complementary to the nucleotide sequence shown by the sequence number in the above table under stringent conditions, and the activity of each gene (activity as interleukin 1)
  • mutant genes having activity as CCL1 can also be targeted, and probes can be designed based on the base sequence of such mutant genes.
  • stringent conditions the same conditions as described above can be applied.
  • a plurality of the above-mentioned “probes” are arranged on the base.
  • the substrate on which the probe is arranged is generally called a DNA chip or a DNA microarray. Examples of the substrate include a glass plate, a resin plate, and a silicon plate.
  • a functional group is exposed on the surface of the base by chemically modifying the base in advance, and a probe is placed on the functional group by spotting (spotting method), or photoinduced to a silicon base.
  • spotting method spotting method
  • a method of synthesizing probes sequentially by a solid layer reaction one photolithographic method is known.
  • a microarray having a plurality of through-holes and holding a gel in these through-holes (hereinafter referred to as a through-hole array) is also preferably used.
  • the through-hole array can be manufactured, for example, by the following method (see Japanese Patent Application Laid-Open No. 2 00-78 998).
  • a hollow fiber type microarray can be exemplified (JP 2000-245461 A, JP 2001-239594 A, JP 2003-344400 A). No. publication). Since the hollow fiber type microarray can realize a highly accurate and inexpensive microarray, it is preferable to use the hollow fiber type microarray in the present invention.
  • the hollow fiber type microarray can be manufactured, for example, by sequentially performing the following steps.
  • the fibers are aligned by passing the hollow fibers through the holes of a plurality of jigs having holes of the same pattern as the target arrangement pattern.
  • the fiber bundle is fixed by filling the matrix resin between the fibers.
  • a block (array) in which the fiber bundle is fixed by the resin can be obtained.
  • a gel precursor solution containing a probe is introduced into the hollow part of each fiber, a gelation reaction is performed in the hollow part, and the probe is fixed to the gel.
  • This cutting means slicing the block in a direction crossing the longitudinal direction, and thereby, a slice of the array can be obtained.
  • a probe is fixed to the fiber portion of the flakes.
  • the method for producing the fiber type microarray is described in JP-A-2001-239594, and the fiber type microarray can be produced by referring to the publication. Further, it is preferable that a gel such as acrylamide is held in the hollow portion, and the oligonucleotide (probe) is fixed (held) to the gel. Such fixation allows the probes to be three-dimensionally arranged in the gel and maintain a three-dimensional structure. As a result, the detection efficiency is increased compared to a planar microarray in which a probe is bonded to a glass slide coated on the surface, and high-sensitivity and high-reproducibility inspection can be performed.
  • the number of types of probes arranged in the microarray is preferably not more than 100, more preferably not more than 500, more preferably not more than 2500 on one (middle) microarray.
  • the type of probe is distinguished by the base sequence. Therefore, even if the probes are derived from the same gene, even if one base sequence is different, it is specified as a different type.
  • the present invention is a method for testing allergenicity and z or antiallergenicity of a test substance, comprising the following steps.
  • oligonucleotide or microarray A step of bringing the biological substance or its preparation into contact with the above-mentioned oligonucleotide or microarray.
  • Allergenic refers to the property of a food that functions as an allergen when ingested, resulting in an allergic response.
  • Allergen refers to a causative substance that causes an allergic reaction. Allergens are processed by macrophages and presented to antigen recognition T cells to induce antigen-specific T cells and antibody production. The type of cell or antibody to be induced and the symptoms of allergic reaction differ depending on the condition of the sensitized living body (eg, genetic factor, nutritional state) or the allergen entry route.
  • Anti-allergic means the property of suppressing the allergic response. This allergic response involves a number of steps, which may be able to suppress allergies at each step. Therefore, an antiallergic substance may function in any step of allergy development as long as it suppresses the allergic response.
  • examples of the food containing antiallergic ingredients include tea, and examples of the ingredients having antiallergenicity include green tea catechins.
  • a test substance is administered to or contacted with a laboratory animal and / or a cell is contacted.
  • Test substance is a substance to be examined. For example, food, a homogenized solution thereof, and a crude product thereof can be mentioned.
  • test substance test food
  • the mode of contacting with a cell means, for example, adding a candidate substance into a cell culture medium or culturing cells together with the candidate substance. A short period of several days may be sufficient as the test substance is administered to experimental animals.
  • cells are not limited to the cell cycle period, and any phase of cells may be used.
  • the test substance When the test substance is administered parenterally (especially during injection), the test substance is a liquid.
  • liquid culture When the test substance is brought into contact with cells, liquid culture is usually performed. Therefore, when liquid foods such as drinks are used as test substances, they can be used as liquids or diluted in an appropriate buffer solution, and when solids are used as test substances, they can be used in a suitable buffer or culture medium. And then diluted to a concentration suitable for culture or administration.
  • cell types include allergy-related cells such as B cells, T cells, basophils, macrophages, and mast cells.
  • the expression of all or part of the IL-1 Q! Gene, IL-liS gene, IL-2 gene, IL-18 gene, CCL2 gene and TNFa gene is enhanced.
  • whether or not the enhancement of all or part of the above genes is suppressed by administering or contacting a test substance having an allergic effect to experimental animals and / or contacting them with cells. Is analyzed.
  • biological substances are extracted from laboratory animals and Z or cells.
  • Biologically related substances are DNA, RNA, protein, peptide, peptide nucleic acid, lipid, antibody and the like. Those extraction methods are appropriately selected by those skilled in the art.
  • the extracted biological substance or its preparation is brought into contact with the above-described probe or microarray.
  • the method of the present invention provides a probe or its probe after the contact.
  • “Preparations” are, for example, extracted biological substances labeled with RI, fluorescence, etc., nucleic acids amplified using PCR, T7 amplification, fragmented nucleic acids, and the like. The reactivity with the probe can be observed by bringing the biological substance or preparation thereof into contact with the gene or microarray.
  • Reaction conditions are appropriately selected depending on the type of biological substance to be used, the type of probe, and the like.
  • the reactivity between the biological substance and the probe is evaluated by an appropriate detection means.
  • the allergenicity and / or antiallergenicity is evaluated by measuring the fluorescence intensity with a fluorescence microscope.
  • evaluation means, for example, quantifying the expression level from the measured fluorescence intensity and comparing it with the control group to determine the type of the specific gene expressed when exposed to the test substance, and its expression level. It is to identify.
  • the probe of the present invention can be used to determine allergenicity and / or antiallergenicity against type I allergy or type IV allergy.
  • the probe or microarray of the present invention it is possible to easily determine and examine allergenicity, so that it is easy to properly display foods with allergenicity. This helps to build a healthy diet, prevent allergies, and build confidence in food companies, leading to industrial progress.
  • the present invention makes it possible to easily determine and test antiallergic properties, it can be used for screening candidate substances at the research and development sites for pharmaceuticals and foods. Therefore, the probe, microarray and method of the present invention are extremely useful in that the present invention can increase the efficiency or speed of the development of new pharmaceuticals and new functional foods, reduce development risks, and reduce development costs. .
  • the present invention provides an allergenic and non-allergic anti-allergic kit comprising the probe or microarray.
  • the kit of the present invention may contain other components in addition to the microarray.
  • other components include fluorescent reagents, restriction enzymes, various buffers, sterilized water, detergents, surfactants, and experimental operation manuals (instructions).
  • a through-hole type microarray was selected as the microarray to be used in the experiment, and a 5'-terminal vinylated nucleic acid molecule (65mer) having the sequence information shown in Table 1 was used as the probe nucleic acid molecule for placement on the DNA microarray. It was.
  • No. 104 is a probe corresponding to IL-1 gene
  • No. 105 is IL-1 ⁇ 6 gene
  • No. 107 is IL-2 gene
  • No. 103 is IL-18 gene
  • No. .19 is the CCL2 gene
  • No.24 is the CCL3 gene
  • No.25 is the CCL4 gene
  • No.26 is the CCL5 gene
  • No.208 is the probe corresponding to the TNF gene.
  • LPS Lipopolysaccharide
  • TPCK Mode: 1J- (tosylamido-2-phenyl) ethyl chloromethyl
  • RAW246.7 cells cultured cells derived from mouse macrophages were used as cells.
  • RAW246.7 cells were cultured for 6 hours in the presence of LPS stimulation alone and in the presence of LPS stimulation and TPCKlOOpmol, and the cells were collected and total RNA was extracted. Total RNA was extracted using RNeasy MinElute Kit (QIAGEN).
  • the single stimulation group for LPS stimulation was the allergy induction model group, and the culture group in the presence of TPCK was the allergy suppression model group.
  • Biotin-labeled aRNA to be hybridized to the microarray of the present invention was prepared and subjected to hybridization.
  • Biotin-labeled aRNA was prepared according to a predetermined method using Message Amp II Biotin Kit (Ambion).
  • a hybridization solution was prepared as follows.
  • Biotin-labeled aRNA is suspended in 15 g Z lSO 1 in a hybridization buffer solution (0.12M TNT solution), and a hybridization reaction is carried out at 65 for 17 hours using a microarray (GenoPa) manufactured by Mitsubishi Rayon. The After the reaction, the chip is washed at 65 ° C using a washing buffer solution (0.12M TNT solution). Two 20 minute washes were performed, and a final wash with 2 x SSC for 10 minutes. Chip detection was performed using a detector manufactured by Mitsubishi Rayon.
  • Fluorescent labeling was performed by immersing the microarray for 30 minutes in 5 ml of a 1: 500 dilution of Cy5-Streptavdin (GE Healthcare) dissolved in lmg Z ml with distilled water. . After immersion, the microarray was washed 4 times for 5 minutes with 5 ml of 0.12M TNT solution, and the fluorescence intensity was measured with a DNA chip detector manufactured by Mitsubishi Rayon.
  • the measured fluorescence intensity was corrected based on the fluorescence intensity of each group GAPD gene (NM_008084), and the fluorescence intensity of the target gene in each group was compared.
  • the GAPD gene (LPS single stimulation group fluorescence intensity value / TPCK stimulation group fluorescence intensity value) was (1 4 7 1 5 7/1 4 5 9 5 3), and the ratio was 1.01. . Therefore, when the relative expression difference of GAPD was expressed in log (bottom 2), it was 0, and the relative expression difference was calculated for the results of other genes (Fig. 1).
  • LPS Lipopolysaccharide: Gram-negative bacterium-derived lipopolysaccharide
  • OVA egg white albumin: ovalbumin
  • IL-la.IL-l S, CCL2, CCL3, CCL4, CCL5, TNFo!
  • IL-2 and IL-18 were found to be enhanced when OVA was administered (Fig. 2).
  • LPS Lipopolysaccharide: lipopolysaccharide derived from gram-negative bacteria
  • PMA (12-O-tetradecanoylp orbol 13-acetate) were used as substances capable of inducing an allergic state.
  • LPS induces an inflammatory response, in other words, it can induce a characteristic response to type V allergy.
  • PMA also promotes secretion of various site forces and induces a reaction different from type IV allergy.
  • These substances were added to mouse cells and stimulated under the following conditions. Total UNA was extracted from these cell groups, and mRNA expression of each gene was measured with a microarray. The results are shown in FIG. 4 and FIG.
  • RNA is extracted by collecting the cells after 6 hours
  • an oligonucleotide probe and a microarray that can efficiently and effectively determine the presence or absence of an allergy onset or a suppressive action due to food components.
  • the oligonucleotide probe and microarray of the present invention are easy to determine or test the allergenicity of unknown food ingredients, or to determine allergenic or antiallergic effects in the development stage of functional food ingredients. It can also be used as a tool. Sequence Listing Free Text SEQ ID NOs: 1-2 2 1: Synthetic DNA

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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

明 細 書 ァレルゲン性又は抗ァレルギ一性判定用プローブ 技術分野  Description Allergenicity or antiallergenicity determination probe Technical Field
本発明は、 食品成分のアレルゲン性及び 又はアレルギー性を判定するために 用いられるオリゴヌクレオチドプローブ及びそれらが配置されたマイクロアレ ィに関する。 背景技術  The present invention relates to oligonucleotide probes used for determining the allergenicity and / or allergenicity of food ingredients, and microarrays on which they are arranged. Background art
近年、 食経験のない作物の食品への導入や、 特定食品の消費量の増加など、 食 生活の変化に伴い、 食物アレルギーの危険性が増大しつつある。 現在、 日本人の 1〜3 %程度は、 食物アレルギーの患者であると推定されている （国民生活セン ター）。 また、 食の安全性及び信頼性に関する消費者のニーズは年々拡大してき ており、 特にアレルゲンの含有に関しては、 特定物質について加工食品の原材料 表示が義務づけられている。 このように、 食品中のアレルゲンと安全性の関係は 深刻化しつつあるのが現状であり、 未知のアレルゲンに対する簡便で再現性の良 いアレルゲン性の判定法の開発が必要とされている。  In recent years, the risk of food allergies has been increasing due to changes in dietary habits, such as the introduction of crops with no food experience into foods and increased consumption of specific foods. Currently, it is estimated that about 1-3% of Japanese people are food allergic patients (National Life Center). In addition, consumer needs for food safety and reliability are increasing year by year. In particular, regarding the inclusion of allergens, labeling of processed food ingredients for specific substances is required. In this way, the relationship between allergens in foods and safety is becoming increasingly serious, and there is a need to develop a simple and highly reproducible allergenicity judgment method for unknown allergens.
また、 アレルギー患者の増大に伴い、 食品に含まれる成分には、 安全性の高い アレルギー抑制又は緩和作用を有するものであることが求められており、 食品の ァレルギ一惹起作用を科学的に解明し、 ァレルギ一を起こさない食品の開発を目 指すことも重要である。  In addition, along with the increase in allergic patients, ingredients in foods are required to have highly safe allergy suppression or alleviation action, and scientifically elucidated the allergic action of foods. It is also important to aim to develop foods that do not cause allergies.
従来、 アレルゲン性の判定法としては ELISA法が用いられていた。 しかしな がら、 この方法は既知のアレルゲン性物質を検出するに止まり、 未知の物質につ いてはアレルゲンを検出することができない。 このため、 食されたことが全くな い未知の作物のアレルゲン性を判定するには、 長期間の動物への投与により推測 する以外に方法が無く、 簡便なアレルゲン性の判定法はこれまでに知られていな レ^ また、 食品成分のアレルゲン性ゃ抗アレルギー作用を判定する場合、 種々の 食品の複合的効果、 すなわち複数の食品を併用して食するとアレルゲン性が変化 するという効果について考慮する必要があるが、 これを ELISA法で行うことは 実質上困難であり、 このような変化を確認するには、 複数の遺伝子の発現挙動を 解析することで初めて可能となると考えられる。 Conventionally, ELISA has been used as a method for determining allergenicity. However, this method only detects known allergenic substances and cannot detect allergens for unknown substances. For this reason, there is no method to determine the allergenicity of an unknown crop that has never been eaten, other than to estimate it by long-term administration to animals. Not known les ^ In addition, when determining allergenic anti-allergic effects of food ingredients, It is necessary to consider the combined effects of foods, that is, the effect that allergenicity changes when multiple foods are used in combination, but this is practically difficult to perform by ELISA, and such changes are This can only be confirmed by analyzing the expression behavior of multiple genes.
近年、 ヒトゲノム解読を初めとする生物ゲノム情報に関する研究の進展は著し く、 特に医療の分野ではこの情報を応用して実際の診断又は治療活動に活かすベ く、 ポストゲノム研究が極めて盛んに行われている。 このポストゲノム研究やそ の応用分野において、 極めて有効なツールとして認識されているものは、 DNA マイクロアレイ （DNAチップとも呼ぶ） である。 DNAマイクロアレイとは、 微 少な面積を有する区画に多数の遺伝子断片を貼り付けたツールであり、 核酸同士 のハイブリダィゼーシヨン反応によってある試料中の遺伝子断片の有無や量等 を検出することで、 複数の遺伝子の発現挙動を簡便に解析することが可能である。  In recent years, research on biological genome information, including human genome decoding, has made significant progress, and post-genomic research has been actively carried out, particularly in the medical field, to apply this information to actual diagnosis or treatment activities. It has been broken. A DNA microarray (also called a DNA chip) is recognized as an extremely effective tool in this post-genome research and its application fields. A DNA microarray is a tool in which a large number of gene fragments are attached to a section having a small area. By detecting the presence or amount of gene fragments in a sample by a hybridization reaction between nucleic acids. It is possible to easily analyze the expression behavior of multiple genes.
DNAマイクロアレイは、 現在、 フォトリソグラフィ一法やスポッティング法 により製造されたものが主に研究用ツールとして使用されているが、 繊維と樹脂 の技術を応用した本出願人独自のプラットフォームとして 「繊維型 DNAマイク ロアレイ （商品名ジエノパール）」 （文献 1〜 3 ) が開発され上市されている (URL:http：〃 www.mr co.jp/genome/)。 この繊維型 DNAマイクロアレイは、 独 自の製造方法の採用により、 高精度の DNAマイクロアレイを低コストで大量生 産できる点が特徴である。 また、 アレイの製造工程における操作は熟練を必要と しないため、 検査従事者の個人差などによる検査結果のばらつき等がなく、 さら には実験動物毎の個体差を加味する必要がない。 したがって、 上記繊維型 DNA マイクロアレイを使用すると正確なデータを簡便に取得することができる。  Currently, DNA microarrays manufactured by a single photolithography method or spotting method are mainly used as a research tool. Microarray (trade name Dienopearl) "(references 1-3) has been developed and marketed (URL: http: 〃 www.mr co.jp/genome/). This fiber-type DNA microarray is characterized by the ability to mass-produce high-precision DNA microarrays at low cost by adopting an original manufacturing method. In addition, since the operation in the array manufacturing process does not require skill, there is no variation in test results due to individual differences among test personnel, and there is no need to take into account individual differences among laboratory animals. Therefore, accurate data can be easily obtained by using the fiber DNA microarray.
ところで、 アレルギーの発症や抑制に関わる研究として、 これまでに、 アレル ギー抑制効果に関わる炎症性サイトカイン産生抑制効果や脱顆粒抑制効果等を 示す食品成分を明らかにすると共に、 その詳細な作用機構の検討及び解明が進め られている （文献 4 )。 その結果、 アレルギーの発症や抑制に関連する遺伝子に 関する基礎的な知見が得られている。  By the way, as research related to the onset and suppression of allergies, we have clarified food ingredients that show inflammatory cytokine production suppression effects and degranulation suppression effects related to allergy suppression effects, and detailed action mechanisms of them. Investigation and elucidation are underway (Reference 4). As a result, basic knowledge about genes related to the onset and suppression of allergies has been obtained.
近年、 遺伝子組み換え食品や食に供されたことがない物質の中には、 アレルゲ ン性ゃ抗アレルギー性が未知のものが多い。 しかしながら、 これらの食品等は市 場に多く流通しているのが現状である。 従って、 食品のアレルゲン性及び抗ァレ ルギ一性を知ることは、 アレルギー体質を有する者にとって極めて重要である。 これらのアレルゲン性等を有するか否かは、 数ケ月以上にわたりアレルギー性未 知の食品等を実験動物に長期継続投与して、 アレルギー症状を観察することによ つて判断される。 しかし、 このように時間を費やす検討を行っても、 得られる結 果は推測の域を超えなかつた。 参照文献 In recent years, genetically modified foods and substances that have never been used in food include allergic Many of them have unknown antiallergic properties. However, the current situation is that many of these foods are distributed in the market. Therefore, knowing the allergenicity and antiallergenicity of food is extremely important for those with allergic predisposition. Whether or not they have these allergenic properties is determined by observing allergic symptoms by continually administering foods that are not known to be allergic to experimental animals for several months or longer. However, even with this time-consuming study, the results obtained did not exceed the speculation. References
1. 特許第 3515470号明細書  1. Patent No. 3515470
2. 特許第 3510882号明細書  2. Patent 3510882 specification
3. 特許第 3515570号明細書  3. Patent No. 3515570
4. Kobori et al. Cell Death Differ., 11(1) 123-130(2004) 発明の開示  4. Kobori et al. Cell Death Differ., 11 (1) 123-130 (2004) Disclosure of the Invention
本発明は、 食品成分のアレルゲン性又は抗アレルギー性を判定する簡便かつ再 現性の高い遺伝子群の提供及びそれらが配置された DNAマイクロアレイを提供 することを目的とする。 本発明者は、 上記課題を解決するため鋭意研究を行った結果、 食品成分による アレルギーの発症又は抑制に関する知見と、 DNAマイクロアレイ技術とを組み 合わせることで、 食品成分のアレルゲン又は抗アレルギー性を再現性良く判定す る効率的で簡便かつ再現性の高い遺伝子群を見出した。 また、 それらが配置され た DNAマイクロアレイを開発することに成功し、 本発明を完成した。  An object of the present invention is to provide a simple and highly reproducible gene group for determining the allergenicity or antiallergenicity of food components and a DNA microarray in which they are arranged. As a result of earnest research to solve the above problems, the present inventor has reproduced allergens or antiallergenicity of food ingredients by combining knowledge about the onset or suppression of allergies caused by food ingredients with DNA microarray technology. An efficient, simple and highly reproducible gene group was determined. In addition, the present inventors have succeeded in developing a DNA microarray in which they are arranged, thereby completing the present invention.
すなわち、 本発明は食物アレルギー応答に関連する遺伝子の塩基配列のうち、 少なくとも一部の塩基配列にハイプリダイズすることができる塩基配列を含む、 アレルゲン性及び Z又は抗アレルギー性判定用オリゴヌクレオチドプロープで ある。 食物アレルギー応答に関連する遺伝子としては、 例えば IL-l a遺伝子、 IL-1 13 遺伝子、 IL-2遺伝子、 IL-18遺伝子、 CCL2遺伝子、 CCL3遺伝子、 CCL4遺伝 子、 CCL 5遺伝子及び TNF o!遺伝子からなる群から選ばれる少なくとも 1つが 挙げられる。 また、 本発明において、 アレルゲン性及びノ又は抗アレルギー性判 定は、 例えば I型アレルギ一又は IV型アレルギーに対するものである。 That is, the present invention is an oligonucleotide probe for determining allergenicity and Z or antiallergenicity, comprising a nucleotide sequence that can be hybridized to at least a part of the nucleotide sequence of a gene related to a food allergy response. is there. Examples of genes related to food allergy response include IL-la gene, IL-1 13 gene, IL-2 gene, IL-18 gene, CCL2 gene, CCL3 gene, CCL4 gene, CCL 5 gene and TNF o! And at least one selected from the group consisting of genes. In the present invention, the allergenicity and anti-allergic determination is for, for example, type I allergy or type IV allergy.
さらに、 本発明は、 上記オリゴヌクレオチドプローブが基盤に配置された、 ァ レルゲン性及び Z又は抗ァレルギ一性判定用マイクロアレイである。  Furthermore, the present invention is a microarray for determining allergenicity and Z or antiallergenicity, wherein the oligonucleotide probe is arranged on a base.
さらに、 本発明は、 複数の貫通孔を有し、 それら貫通孔にゲルが保持されてい るマイクロアレイであって、 前記ゲルに上記オリゴヌクレオチドプロ一ブが保持 されている、 アレルゲン性及び Z又は抗アレルギー性判定用マイクロアレイであ る。  Furthermore, the present invention provides a microarray having a plurality of through-holes and a gel held in the through-holes, wherein the oligonucleotide probe is held in the gel. It is a microarray for determining allergenicity.
本発明のマイクロアレイにおいて、 オリゴヌクレオチドプロ一ブは、 好まし くは 1 0 0 0種類以下である。  In the microarray of the present invention, the number of oligonucleotide probes is preferably 100 or less.
さらに、 本発明は、 以下の工程を含む、 被験物質のアレルゲン性及び/又は抗 アレルギー性の判定方法である：  Furthermore, the present invention is a method for determining the allergenicity and / or antiallergenicity of a test substance, comprising the following steps:
( 1 ) 被験物質を実験動物に投与し若しくは接触させ、 及び Z又は被験物質 を細胞に接触させる工程、  (1) The step of administering or contacting a test substance to a laboratory animal, and contacting Z or the test substance to a cell,
( 2 ) 実験動物及び Z又は細胞から生体関連物質を抽出する工程、 並びに (2) A process for extracting biological substances from laboratory animals and Z or cells, and
( 3 ) 前記生体関連物質又はその調製物を、 前記オリゴヌクレオチドプロ一 ブ又は前記マイクロアレイに接触させる工程。 以下に本発明の実施の形態について説明する。 以下の実施形態は、 本発明を説 明するための単なる例示であって、 本発明をこの実施形態にのみ限定することは 意図されない。 本発明は、 その要旨を逸脱しない限り、 様々な形態で実施するこ とが可能である。 (3) A step of bringing the biological substance or its preparation into contact with the oligonucleotide probe or the microarray. Embodiments of the present invention will be described below. The following embodiment is merely an example for explaining the present invention, and the present invention is not intended to be limited to this embodiment. The present invention can be implemented in various forms without departing from the gist thereof.
なお、 本明細書において引用した全ての刊行物、 例えば、 先行技術文献および 公開公報、 特許公報その他の特許文献は、 その全体が本明細書において参照とし て組み込まれる。 本明細書は、 本願優先権主張の基礎となる特願 2006-327852号明細書の内容を 包含する。 本発明により、 食品成分によるアレルギー発症又は抑制作用の有無を効率的か つ効果的に判定をすることができるオリゴヌクレオチドプロ一ブ及びマイク口 アレイが提供される。 また、 本発明のオリゴヌクレオチドプローブ及びマイクロ アレイは、 未知の食品成分のアレルゲン性を判定又は検査したり、 機能性食品成 分の開発段階におけるアレルゲン性又は抗アレルギー性作用を判定するための 簡便なツールとしても使用することができる。 All publications cited in the present specification, for example, prior art documents and published publications, patent publications and other patent documents, are incorporated herein by reference in their entirety. This specification includes the contents of Japanese Patent Application No. 2006-327852, which is the basis for claiming priority of the present application. INDUSTRIAL APPLICABILITY According to the present invention, there are provided an oligonucleotide probe and a microphone mouth array capable of efficiently and effectively determining the presence or absence of an allergy onset or an inhibitory action due to food components. In addition, the oligonucleotide probe and microarray of the present invention are easy to determine or test the allergenicity of unknown food ingredients, or to determine allergenic or antiallergic effects in the development stage of functional food ingredients. It can also be used as a tool.
本発明において、 食品成分のアレルゲン性及びノ又は杭アレルギー性の判定方 法は、 数日から数週間程度の短い期間にアレルギー性の未知の食品等を投与した 実験動物又は培養細胞から核酸を抽出し、 これを本発明で見出した遺伝子群やそ れらを配置したマイクロアレイに接触させることを特徴とする。 本発明の方法に より、 アレルギー性等の評価の短時間化、 省力化、 結果の安定化が実現し、 さら に、 数種の食品の複合効果を判定することができる。 図面の簡単な説明  In the present invention, the method for determining the allergenicity of food ingredients and the allergenicity of piles or piles is based on the extraction of nucleic acids from experimental animals or cultured cells administered with unknown allergenic foods in a short period of time from several days to several weeks This is characterized in that it is brought into contact with a gene group found in the present invention or a microarray on which these genes are arranged. According to the method of the present invention, it is possible to shorten the evaluation of allergy and the like, save labor, and stabilize the results. Further, the combined effect of several kinds of foods can be determined. Brief Description of Drawings
図 1は、 TPCKで発現抑制された遺伝子群を示す図である。  FIG. 1 is a diagram showing a group of genes whose expression was suppressed by TPCK.
図 2は、 LPS及び OVAを用いてアレルギー惹起の様子を判定した結果を示す 図である。  FIG. 2 is a diagram showing the results of determining the state of allergenicity using LPS and OVA.
図 3は、 LPS及び OVAを用いてアレルギー惹起の様子を判定した結果を示す 図である。  FIG. 3 is a diagram showing the results of determining the state of allergenicity using LPS and OVA.
図 4は、 LPS及び PMAを用いてアレルギー惹起の様子を判定した結果を示す 図である。  FIG. 4 is a diagram showing the results of determining the state of allergenicity using LPS and PMA.
図 5は、 LPS及び PMAを用いてアレルギー惹起の様子を判定した結果を示す 図である。 発明を実施するための最良の形態 FIG. 5 is a diagram showing the results of determining the state of allergenicity using LPS and PMA. BEST MODE FOR CARRYING OUT THE INVENTION
本発明は、 アレルゲンを含む食品又はアレルゲンを含むと思われる食品候補物 質を食し、 又はこれら食品等に接触したときに変動する遺伝子に着目することに より完成されたものであり、 これらの遺伝子又はその一部にハィブリダイズする 塩基配列を有するオリゴヌクレオチドプローブが配置されたマイクロアレイに 関するものである。 すなわち、 本発明は、 複数の食物アレルギー応答に関連する ォリゴヌクレオチドプロ一ブが配置されたマイクロアレイである。  The present invention has been completed by paying attention to genes that change when eating food products containing allergens or food candidate substances that are thought to contain allergens, or coming into contact with these foods. Alternatively, the present invention relates to a microarray in which oligonucleotide probes having a base sequence that hybridizes to a part thereof are arranged. That is, the present invention is a microarray in which oligonucleotide probes related to a plurality of food allergic responses are arranged.
「食物アレルギー応答」 とは、 経口摂取若しくは経鼻吸入された食物成分、 又 は皮膚に接触した食物成分が原因となって過剰な免疫応答を起こし、 食物を摂取 等した個体において種々の体調の異常を誘起する障害反応をいう。 食物アレルギ —には、 （a) アレルゲンに特異的な IgE抗体により即時型の症状を引き起こすも の、 あるいは (b)アレルゲン特異的炎症性 T細胞、好酸球又はマクロファージによ る遅延型の腫脹又は組織破壌を起こすものがある。 食物アレルギーは、 消化管、 気道、 皮膚に発症し易く、 特にアトピー性皮廇炎と深く関連すると考えられてい る。 ここで、 アレルゲンを含む食物としては、 例えば卵、 乳、 食肉 （牛肉、 鶏肉、 豚肉等)、 穀物 （大豆、 米、 小麦、 ソバ、 ゴマ、 落花生等)、 やまいも、 魚介類 （あ わび、 いか、 いくら、 えび、 かに、 さけ、 さば等）、 果物 （オレンジ、 キウイフ ルーツ、 もも、 りんご、 いちじく等） などが挙げられる。 “Food allergic response” refers to food components that are ingested or nasally inhaled, or that cause excessive immune responses due to food components that come into contact with the skin, and have various physical conditions in individuals who have consumed food. It refers to a disordered reaction that induces abnormalities. 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 May cause swelling or tissue destruction. Food allergies tend to occur in the gastrointestinal tract, respiratory tract, and skin, and are thought to be closely related to atopic dermatitis. Here, examples of foods containing allergens include eggs, milk, meat (beef, chicken, pork, etc.), cereals (soybean, rice, wheat, buckwheat, sesame, peanuts, etc.), sweet potatoes, seafood (abalone, squid) , How much, shrimp, crab, salmon, mackerel, etc.), fruits (orange, kiwi roots, peaches, apples, figs, etc.).
また、 アレルゲンとしては、 例えば、 卵においてはオボアルブミン、 オボムコ イドなど、 乳においてはカゼイン、 ；6ラクトグロブリンなど、 小麦においてはグ リアジン、 aアミラーゼインヒビ夕一など、 落花生においては 2 Sアルブミンな ど、 魚介類においてはパルプアルブミン、 トロポミオシンなどが挙げられる。 本発明において、 「オリゴヌクレオチドプローブ」 とは、 アレルギー応答の促 進又は抑制に関連して発現量が変化する遺伝子を検出するための探査針となる ものをいう。 このようなプローブとして、 例えばアレルゲンを有する食物の摂取 によって免疫応答が生じ、 アレルギー物質が産生されるときに発現量が変化する 遺伝子、 あるいはアレルギー反応を抑えるために抑制物質が産生されるときに発 現量が変化する遺伝子を検出するためのオリゴヌクレオチドが挙げられる （以下、 プロープという）。 Allergens include, for example, ovalbumin and ovomucoid in eggs, casein in milk; 6 lactoglobulin, gliadin, a-amylase inhibitor in wheat, and 2S albumin in peanuts. However, in seafood, pulp albumin, tropomyosin and the like can be mentioned. In the present invention, “oligonucleotide probe” refers to a probe that serves as a probe for detecting a gene whose expression level changes in association with promotion or suppression of an allergic response. As such a probe, for example, when an allergic substance is produced by ingesting an allergen-containing food, an expression level changes, or when an inhibitory substance is produced to suppress an allergic reaction. Examples include oligonucleotides for detecting genes whose actual amount changes (hereinafter, Called a probe).
検出の目的となる遺伝子は、 アレルギー応答の促進又は抑制に関連して発現量 が変化する遺伝子等であれば特に限定されるものではないが、 各種サイトカイン、 ヒスタミン等に関連する遺伝子群であることが好ましい。 とりわけ、 食物アレル ギ一として代表的な I型アレルギー及び IV型アレルギーを判別するのに有用な 最小単位の遺伝子群である。  The gene to be detected is not particularly limited as long as it is a gene whose expression level changes in relation to the promotion or suppression of allergic response, but it is a gene group related to various cytokines, histamine, etc. Is preferred. In particular, it is a gene group of the smallest unit useful for discriminating type I allergy and type IV allergy, which is a typical food allergy.
ここで、 「I型アレルギー」 とは、 抗原 （アレルゲン） との接触後即座に反応が 起こるアレルギーである。 IgEクラスの抗体は、 その Fc部で肥満細胞上の Fe s レセプ夕一に結合する。 これに抗原が結合すると、 Fcレセプ夕一同士が抗原分子 を通して結合する。 これにより、 肥満細胞からヒスタミンが細胞外に放出され、 ロイコトリェン、 血小板活性化因子などのケミカルメデイエ一夕一が合成及び放 出される。 ケミカルメディエー夕一は、 血管透過性亢進、 平滑筋収縮、 白血球走 化などをもたらし、 その結果組織反応によって症状が発現する。  Here, “type I allergy” is an allergy that reacts immediately after contact with an antigen (allergen). IgE class antibodies bind to Fe s receptors on mast cells at their Fc part. When an antigen binds to this, the Fc receptors bind through the antigen molecule. As a result, histamine is released from mast cells to the outside of the cell, and chemical media such as leukotriene and platelet activating factor are synthesized and released. Chemical mediator Yui leads to increased vascular permeability, smooth muscle contraction, leukocyte chemotaxis, and as a result, symptoms appear due to tissue reaction.
「IV型アレルギー」 とは、 抗体の関与なしに抗原と T細胞との反応によって もたらされる組織傷害をいう。 T細胞がインターロイキン （IL) 等のリンホカイ ンを産生し、 これによりマクロファージゃ好中球の浸潤、 血管透過性亢進による 血漿の滲出、 繊維芽細胞の増殖などがもたらされる。  “Type IV allergy” refers to tissue injury caused by the reaction between an antigen and T cells without antibody involvement. T cells produce lymphokines such as interleukin (IL), which leads to macrophage infiltration of neutrophils, plasma exudation due to increased vascular permeability, and proliferation of fibroblasts.
本発明においては、 このようなアレルギーに関連する遺伝子群を対象とする。 これらの遺伝子群としては、 IL-1 (インターロイキン） α遺伝子、 IL-l jS遺伝子、 IL-2遺伝子、 IL-4遺伝子、 IL-8遺伝子、 IL-10遺伝子、 IL-18遺伝子、 CCL2 (ケ モカイン） 遺伝子、 CCL3遺伝子、 CCL4遺伝子、 CCL 5遺伝子、 PTGS2 (プロ スタグランジン—エンドペルォキシド合成酵素 2 ) および TNF (腫瘍壊死因子） ひ遺伝子が好ましく、 より好ましくは、 IL-l o!遺伝子、 IL-l j6遺伝子、 IL-2遺伝 子、 EL-18遺伝子、 COL2遺伝子、 CC 3遺伝子、 CCL4遺伝子、 CC 5遺伝子 及び TNFひ遺伝子である。 これらの遺伝子は、その 1種又は複数種を組み合わせ て用いることができる。 好ましくは、 これらの遺伝子すベての挙動を観察し判定 に使用する。  In the present invention, the gene group related to such allergy is the subject. These genes include IL-1 (interleukin) α gene, IL-l jS gene, IL-2 gene, IL-4 gene, IL-8 gene, IL-10 gene, IL-18 gene, CCL2 ( Chemokines) genes, CCL3 genes, CCL4 genes, CCL 5 genes, PTGS2 (prostaglandin-endoperoxide synthase 2) and TNF (tumor necrosis factor) genes are preferred, more preferably IL-lo! Genes IL-6 gene, IL-2 gene, EL-18 gene, COL2 gene, CC3 gene, CCL4 gene, CC5 gene and TNF gene. These genes can be used alone or in combination. Preferably, the behavior of all these genes is observed and used for judgment.
本発明においてプローブとして使用されるオリゴヌクレオチドは、 これら遺伝 子の塩基配列のうち、 少なくとも一部の塩基配列とハイブリダィズすることがで きるものである。 すなわち、 本発明のプローブは、 上記遺伝子の塩基配列に対し て相補的となるように設計され、 被験試料中の遺伝子とのハイプリダイゼ一ショ ンを行なったときにハイブリダィズすることができるものを意味する。 このようなプローブは、 上記遺伝子の c DNA を調製し、 そのままマイクロア レイに配置しても良い。 しかし、タンパク質やペプチドの種類が異なっていても、 それらのタンパク質又はペプチドをコードする DNAの中には塩基配列が類似す る領域が存在することがある。 したがって、 選択したプローブの塩基配列によつ ては、 配列が互いに類似する場合が生じ、 そのような塩基配列では複数の遺伝子 を区別して検出することができない。 そのため、 プローブは、 目的遺伝子に特異 的な塩基配列となるような領域を選択してその領域の塩基配列を設計すること が好ましい。 プローブの設計の際には、 特異的な領域を選択することに加えて、 3末端からの距離が 1 5 0 0塩基以内であり、 Tm (融解温度） が 6 0〜 8 0でで あること、 プローブ中の GC塩基含量が 4 0〜6 0 %、 及び二次構造の形成しや すさを加味した上で設計を行うことが好ましい。 これらの条件については後述す る表 1に示されている。 前記の塩基配列を設計するためには、 配置するプローブの由来となる遺伝子間 において、 塩基配列同士のホモロジ一を既存のプログラムを用いて検索し、 相同 性の低い箇所を選択すればよい。 プローブの塩基配列を設計するに際して選択し 得る相同性は 3 0 %以下であり、 好ましくは 2 5 %以下、 さらに好ましくは 1 0 %以下である、 ここで、 設計されるプローブの長さは 1 0〜2 0 0塩基、 好ま しくは 1 0〜1 0 0塩基である。 プローブは、 通常のオリゴヌクレオチド合成法 を使用して化学合成することにより作製することができる。 そのようなプローブ は例えば、 Probe Quest (登録商標：ダイナコム社製） により設計することがで きる。 The oligonucleotide used as a probe in the present invention has these genetics. Among the child base sequences, those that can hybridize with at least a part of the base sequences. That is, the probe of the present invention means a probe designed so as to be complementary to the base sequence of the gene and capable of hybridizing when performing hybridization with the gene in the test sample. . For such a probe, cDNA of the above gene may be prepared and placed in a microarray as it is. However, even if the types of proteins and peptides are different, there may be a region with a similar base sequence in the DNA encoding the protein or peptide. Therefore, depending on the base sequence of the selected probe, the sequences may be similar to each other, and such a base sequence cannot distinguish and detect a plurality of genes. Therefore, it is preferable that the probe selects a region that has a base sequence specific to the target gene and designs the base sequence of that region. When designing the probe, in addition to selecting a specific region, the distance from the 3 terminal must be within 1 500 bases and the Tm (melting temperature) must be 60 to 80 It is preferable to design in consideration of the GC base content in the probe of 40 to 60% and the ease of secondary structure formation. These conditions are shown in Table 1 below. In order to design the above-mentioned base sequence, it is only necessary to search for homology between base sequences between the genes from which the probes to be arranged are derived using an existing program, and select a portion having low homology. The homology that can be selected when designing the base sequence of the probe is 30% or less, preferably 25% or less, more preferably 10% or less. Here, the length of the designed probe is 1 It is 0 to 200 bases, preferably 10 to 100 bases. The probe can be prepared by chemical synthesis using a normal oligonucleotide synthesis method. Such a probe can be designed by, for example, Probe Quest (registered trademark: manufactured by Dynacom).
プローブは、 上記のとおり試料とのハイプリダイゼーシヨンをおこなったとき に検出目的遺伝子とハイブリダィズすることができるものである。 したがって、 プロ一ブを設計する際に、 ハイブリダィゼーシヨンにおけるストリンジエンシー を考慮する必要がある。 ストリンジエンシーをある程度緊密にすることによって、 遺伝子間で類似する塩基配列領域が存在しても、 他の異なる領域を区別して八ィ ブリダィズすることができる。 また、 遺伝子間の塩基配列がほとんど異なる場合 は、 ストリンジエンシーを緩やかに設定することができる。 When the probe is subjected to hyperprecipitation with the sample as described above It can be hybridized with a target gene for detection. Therefore, when designing a probe, it is necessary to consider stringency in the hybridization. By making stringency close to a certain extent, even if a similar base sequence region exists between genes, other different regions can be distinguished and hybridized. In addition, when the nucleotide sequences between genes are almost different, stringency can be set gently.
このようなストリンジエンシーの条件としては、 例えば緊密条件の場合は 6 5 °C〜6 8 °Cの条件下でのハイブリダィゼ一シヨンであり、 ゆるやかな条件の場 合は 3 7 °Cから 5 5 °Cの条件下でのハイブリダィゼ一シヨンである。 本発明におけるハイブリダィゼ一ション条件において、 ストリンジェントな条 件としては、例えば、 Γ0.12Μ Tris -HCl O.12M NaCl/0.05% Tween-20、 50°C」、 Such stringency conditions include, for example, hybridization under conditions of 65 ° C to 68 ° C under tight conditions, and 37 ° C to 5 ° under mild conditions. Hybridization under conditions of 5 ° C. In the hybridization conditions of the present invention, stringent conditions include, 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」、 よりストリンジェントな条件として は、例えば「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」 等の条件を挙げることができる。 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 For example, `` 0.12M Tris ΗΟΐΖ0.12Μ Ν & /0.05% Tween-20, 65 ° C '', `` 0.12M Tris HC1 / 0.12M NaCl / 0.05% Tween-20, 68 ° C '', `` 0.06M Tris · HC1 / 0.06M NaCl / 0.05% Tween-20, 65 ° C. ”.
より詳細には、プローブを添加して 1時間以上 65°Cに保ってハイプリッド形成 させ、 その後、 0.12M Tris 'HCL/0.12M NaCl/ 05% Tween-20中、 65°Cで 20 分の洗浄を 4回、 最後に、 0.12M Tris-HCl/0.12M NaCl、 65°Cで 10分の洗浄 を 1回行う方法もある。 ハイブリダィゼーシヨン、 あるいは洗浄の際の温度を上 げることにより、 よりストリンジェントな条件を設定することができる。 当業者 であれば、 このようなバッファーの塩濃度、 温度等の条件に加えて、 その他のプ ローブ濃度、 プローブの長さ、 反応時間等の諸条件を加味し、 条件を設定するこ とができる。  More specifically, the probe is added and kept at 65 ° C for 1 hour or longer to form a hybrid, and then washed in 0.12M Tris'HCL / 0.12M NaCl / 05% Tween-20 at 65 ° C for 20 minutes. There is also a method of washing once for 10 minutes at 65 ° C in 0.12M Tris-HCl / 0.12M NaCl. More stringent conditions can be set by raising the temperature during hybridization or washing. A person skilled in the art can set conditions in consideration of other conditions such as probe concentration, probe length, and reaction time in addition to conditions such as salt concentration and temperature of the buffer. it can.
ハイブリダィゼ一シヨン法の詳細な手順については、 「Molecular Cloning, A Laboratory Manual 2nd ed.J (Cold Spring Harbor Press (1989、 、 「Current Protocols in Molecular BiologyJ (John Wiley & Sons (1987-1997)) 等を参照す ることができる。 本発明において、 検出目的遺伝子の塩基配列は、 上記した各遺伝子の塩基配列 そのものである必要はなく、 塩基配列の一部が欠失、 置換、 挿入等により変異が 生じたものであってもよい。 したがって、 検出目的遺伝子は、 上記表に記載の配 列番号により示される塩基配列に相補的な配列と、 ストリンジェントな条件下で ハイブリダィズし、 かつそれぞれの遺伝子の活性 （インタ一ロイキン 1としての 活性、 CCL1としての活性等） を有する変異型遺伝子も対象とすることができ、 プローブは、 このような変異型遺伝子の塩基配列を基礎として設計することもで きる。 「ストリンジェントな条件」 は、 前記と同様の条件を適用することができ る。 上述の 「プローブ」 は、 基盤に複数配置される。 プローブが配置された基盤は 一般的に DNAチップ又は DNAマイクロアレイと称される。 基盤としては、 ガ ラス板、 樹脂板、 シリコン板等が挙げられる。 配置の方法としては、 基盤を予め 化学修飾することにより基盤表面に官能基を露呈させ、 その官能基に対してスポ ッティングによりプローブを配置する方法 （スポッティング法）、 あるいはシリ コン基盤状に光誘起固層反応によって逐次プローブを合成する方法 （フォトリソ グラフィ一法） が知られている。 For details on the hybridization procedure, see `` Molecular Cloning, A Laboratory Manual 2nd ed.J (Cold Spring Harbor Press (1989, `` Current Protocols in Molecular Biology J (John Wiley & Sons (1987-1997)) etc.). Refer Can. In the present invention, the base sequence of the target gene for detection need not be the base sequence itself of each gene described above, and may be a part of the base sequence that has been mutated by deletion, substitution, insertion, or the like. . Therefore, the target gene for detection hybridizes with a sequence complementary to the nucleotide sequence shown by the sequence number in the above table under stringent conditions, and the activity of each gene (activity as interleukin 1) In addition, mutant genes having activity as CCL1) can also be targeted, and probes can be designed based on the base sequence of such mutant genes. As the “stringent conditions”, the same conditions as described above can be applied. A plurality of the above-mentioned “probes” are arranged on the base. The substrate on which the probe is arranged is generally called a DNA chip or a DNA microarray. Examples of the substrate include a glass plate, a resin plate, and a silicon plate. As a placement method, a functional group is exposed on the surface of the base by chemically modifying the base in advance, and a probe is placed on the functional group by spotting (spotting method), or photoinduced to a silicon base. A method of synthesizing probes sequentially by a solid layer reaction (one photolithographic method) is known.
また、 複数の貫通孔を有し、 それら貫通孔にゲルが保持されているマイクロア レイ （以下、 貫通孔アレイと称す。） も好適に使用される。 貫通孔アレイとは、 例えば、 以下の方法により製造できる （特開 2 0 0 0— 7 8 9 9 8号参照）。  Further, a microarray having a plurality of through-holes and holding a gel in these through-holes (hereinafter referred to as a through-hole array) is also preferably used. The through-hole array can be manufactured, for example, by the following method (see Japanese Patent Application Laid-Open No. 2 00-78 998).
( 1 ) 樹脂製のプロックにレーザー等で平行の穴が形成されるように穿つ。 (1) Drill a plastic block so that a parallel hole is formed with a laser or the like.
( 2 ) その穴にプローブを含むゲル前駆体溶液を導入し、穴内でゲル化反応を実 施する。 (2) The gel precursor solution containing the probe is introduced into the hole, and the gelation reaction is performed in the hole.
( 3 ) 樹脂製プロックを穴を横切断する方向で切断を繰り返す。  (3) Repeat cutting of the resin block in the direction of cutting the hole transversely.
また、 貫通孔アレイの好ましい例としては、 中空繊維型マイクロアレイが例示 できる（特開 2000-245461号公報、特開 2001-239594号公報、特開 2003-344400 号公報参照）。 中空繊維型マイクロアレイは高精度で低価格なマイクロアレイを 実現することができるため、 本発明では中空繊維型マイクロアレイを用いること が好ましい。 中空繊維型マイクロアレイは、 例えば以下の工程を順次行うことに より製造できる。 Further, as a preferable example of the through-hole array, a hollow fiber type microarray can be exemplified (JP 2000-245461 A, JP 2001-239594 A, JP 2003-344400 A). No. publication). Since the hollow fiber type microarray can realize a highly accurate and inexpensive microarray, it is preferable to use the hollow fiber type microarray in the present invention. The hollow fiber type microarray can be manufactured, for example, by sequentially performing the following steps.
( 1 ) 中空繊維を目的の配列パターンと同一パターンの孔を有する複数個の治 具の孔に通すことで繊維を整列させる。  (1) The fibers are aligned by passing the hollow fibers through the holes of a plurality of jigs having holes of the same pattern as the target arrangement pattern.
( 2 ) 治具の間隔を目的の幅に広げた後、 繊維間にマトリクス樹脂を充填して 繊維束を固定する。 これにより、 繊維束が樹脂により固定されたプロック （配列 体） を得ることができる。  (2) After expanding the jig spacing to the desired width, the fiber bundle is fixed by filling the matrix resin between the fibers. As a result, a block (array) in which the fiber bundle is fixed by the resin can be obtained.
( 3 ) 各繊維の中空部に、 プローブを含むゲル前駆体溶液を導入し、 中空部内 でゲル化反応を実施し、 ゲルにプローブを固定する。  (3) A gel precursor solution containing a probe is introduced into the hollow part of each fiber, a gelation reaction is performed in the hollow part, and the probe is fixed to the gel.
( 4 ) 繊維の長手方向と交叉する方向で切断する。  (4) Cut in the direction crossing the longitudinal direction of the fiber.
この切断は、 ブロックを長手方向と交叉する方向でスライスすることを意味し、 これにより、 配列体の薄片を得ることができる。 この薄片の繊維部分にプローブ が固定されている。 上記繊維型マイクロアレイの製造方法は、特開 2001-239594号公報に記載され ており、 当該公報を参照することにより繊維型マイクロアレイを製造することが できる。 またその中空部にはアクリルアミド等のゲルが保持され、 オリゴヌクレ ォチド（プローブ） はそのゲルに固定 （保持） されていることが好ましい。 そのよ うな固定により、 プローブはゲル内で 3次元的に配列され、 3次元構造を維持す ることが可能となる。 そのため、 表面をコートしたスライドガラスにプローブを 結合させた平面マイクロアレイに比べて、 検出効率が上昇し、 高感度で高再現性 の検査をすることが可能となる。  This cutting means slicing the block in a direction crossing the longitudinal direction, and thereby, a slice of the array can be obtained. A probe is fixed to the fiber portion of the flakes. The method for producing the fiber type microarray is described in JP-A-2001-239594, and the fiber type microarray can be produced by referring to the publication. Further, it is preferable that a gel such as acrylamide is held in the hollow portion, and the oligonucleotide (probe) is fixed (held) to the gel. Such fixation allows the probes to be three-dimensionally arranged in the gel and maintain a three-dimensional structure. As a result, the detection efficiency is increased compared to a planar microarray in which a probe is bonded to a glass slide coated on the surface, and high-sensitivity and high-reproducibility inspection can be performed.
さらにマイクロアレイに配置されているプローブの種類の数は、 一マイクロア レイ上 （中） に 1 0 0 0種類以下、 好ましくは 5 0 0種類以下、 さらに好ましく は 2 5 0種類以下が好ましい。 このように配置された遺伝子数を制限することに より、 本発明で見出した上述の遺伝子をより高感度で検出することが可能となる。 ここで、 本明細書において、 プローブの種類は塩基配列によって区別される。 し たがって、 同じ遺伝子由来のプローブであっても塩基配列が 1個でも異なれば別 の種類として特定する。 次に被験物質のアレルゲン性及び Z又は抗アレルギー性の検査方法について 説明する。 Further, the number of types of probes arranged in the microarray is preferably not more than 100, more preferably not more than 500, more preferably not more than 2500 on one (middle) microarray. By limiting the number of genes arranged in this way, the above-described genes found in the present invention can be detected with higher sensitivity. Here, in this specification, the type of probe is distinguished by the base sequence. Therefore, even if the probes are derived from the same gene, even if one base sequence is different, it is specified as a different type. Next, the test method for the allergenicity and Z or antiallergenicity of the test substance is explained.
本発明は、 以下の工程を含む、 被験物質のアレルゲン性及び z又は抗アレルギー 性の検査方法である。 The present invention is a method for testing allergenicity and z or antiallergenicity of a test substance, comprising the following steps.
( 1 ) 被験物質を実験動物に投与若しくは接触させ、 及びノ又は被験物質を 細胞に接蝕させる工程、  (1) A step of administering or contacting a test substance to a laboratory animal and caring the test substance or cell into a cell,
( 2 ) 実験動物及び Z又は細胞から生体関連物質を抽出する工程、 並びに (2) A process for extracting biological substances from laboratory animals and Z or cells, and
( 3 ) 前記生体関連物質又はその調製物を、 上述のオリゴヌクレオチド又は マイクロアレイに接触させる工程。 「アレルゲン性」 とは、 摂食時にアレルゲンとして機能し、 結果としてアレル ギー応答を引き起こす食品の性質をいう。 「アレルゲン」 とは、 アレルギー反応 を引き起こす原因物質をいう。 アレルゲンはマクロファージにより処理されて抗 原認識 T細胞に提示され、 抗原特異的な T細胞や抗体産生を誘導する。 なお、 誘 導される細胞又は抗体の種類、 及びアレルギー反応の症状は、 感作される生体側 の条件により （例えば、 遺伝子要因、 栄養状態）、 あるいはアレルゲンの侵入経 路により異なる。 (3) A step of bringing the biological substance or its preparation into contact with the above-mentioned oligonucleotide or microarray. “Allergenic” refers to the property of a food that functions as an allergen when ingested, resulting in an allergic response. “Allergen” refers to a causative substance that causes an allergic reaction. Allergens are processed by macrophages and presented to antigen recognition T cells to induce antigen-specific T cells and antibody production. The type of cell or antibody to be induced and the symptoms of allergic reaction differ depending on the condition of the sensitized living body (eg, genetic factor, nutritional state) or the allergen entry route.
「抗アレルギー性」 とは、 アレルギー応答を抑制する性質を意味する。 このァ レルギ一の応答には数多くのステツプが含まれるため、 それぞれのステップにお いてアレルギーを抑制できる可能性がある。 従って、 抗アレルギー性を有する物 質は、 アレルギー応答を抑制する限り、 アレルギーの発症のいかなるステップに 機能してもよい。 抗アレルギー性を有する成分を含む食物としては、 例えば、 茶 などが挙げられ、 抗アレルギ一性を有する成分としては、 緑茶カテキンなどが挙 げられる。 第一の工程では、 被験物質を実験動物に投与若しくは接触させ、 及び 又は細 胞に接触させる。 「被験物質」 とは、検査の対象となる物質である。例えば食物、 それをホモジネートした溶液、 その粗精製物等が挙げられる。 実験動物に被験物 質 （被験食品） を投与又は接触する形態は限定されるものではなく、 経口投与及 び非経口投与 （注射、 経皮等） のいずれでもよい。 また、 細胞に接触させる態様 は、 例えば細胞培養液中に候補物質を添加すること、 候補物質とともに細胞を培 養することのいずれをも意味するものである。 被験物質を実験動物に投与する期 間は数日間の短期投与でよい。 細胞を使用する場合は、 細胞周期の時期に限定さ れるものではなく、 いずれのフェーズの細胞を使用しても良い。 “Anti-allergic” means the property of suppressing the allergic response. This allergic response involves a number of steps, which may be able to suppress allergies at each step. Therefore, an antiallergic substance may function in any step of allergy development as long as it suppresses the allergic response. Examples of the food containing antiallergic ingredients include tea, and examples of the ingredients having antiallergenicity include green tea catechins. In the first step, a test substance is administered to or contacted with a laboratory animal and / or a cell is contacted. “Test substance” is a substance to be examined. For example, food, a homogenized solution thereof, and a crude product thereof can be mentioned. The form in which the test substance (test food) is administered to or contacted with the experimental animal is not limited, and may be either oral or parenteral (injection, transdermal, etc.). In addition, the mode of contacting with a cell means, for example, adding a candidate substance into a cell culture medium or culturing cells together with the candidate substance. A short period of several days may be sufficient as the test substance is administered to experimental animals. When cells are used, they are not limited to the cell cycle period, and any phase of cells may be used.
被験物質を非経口投与する場合（特に注射のとき）は、被験物質は液体であり、 また、 被験物質を細胞に接触させる場合も、 通常は液体培養を行う。 従って、 飲 料などの液体食品を被験物質とするときは液体のまま又は適当な緩衝液に希釈 して用いることができ、 固体を被験物質とするときは適当な緩衝液又は培養液中 でホモジネ一トし、 その後培養又は投与に適する濃度に希釈することができる。 細胞の種類としては、 例えば、 B細胞、 T細胞、 好塩基球、 マクロファージ、 肥満細胞などのアレルギー関連細胞等が挙げられる。 抗アレルギー性を解析する 場合は、 まず、 アレルギー様の状態を惹起し得る候補物質を実験動物に投与若し くは接触させ、 及び Z又は細胞に接触させる。 この工程により、 例えば IL-1 Q!遺 伝子、 IL-l iS遺伝子、 IL-2遺伝子、 IL-18遺伝子、 CCL2遺伝子および TNF a遺 伝子の全部又は一部の遺伝子の発現が亢進するが、 それに続いてアレルギ一抑制 効果のある被検物質を実験動物に投与若しくは接触させ、 及び/又は細胞に接触 させることで、 上記の遺伝子の全部又は一部において亢進が抑制されるか否かを 解析する。  When the test substance is administered parenterally (especially during injection), the test substance is a liquid. When the test substance is brought into contact with cells, liquid culture is usually performed. Therefore, when liquid foods such as drinks are used as test substances, they can be used as liquids or diluted in an appropriate buffer solution, and when solids are used as test substances, they can be used in a suitable buffer or culture medium. And then diluted to a concentration suitable for culture or administration. Examples of cell types include allergy-related cells such as B cells, T cells, basophils, macrophages, and mast cells. When analyzing antiallergic properties, first, a candidate substance capable of causing an allergic state is administered to or contacted with an experimental animal, and then contacted with Z or cells. Through this process, for example, the expression of all or part of the IL-1 Q! Gene, IL-liS gene, IL-2 gene, IL-18 gene, CCL2 gene and TNFa gene is enhanced. However, whether or not the enhancement of all or part of the above genes is suppressed by administering or contacting a test substance having an allergic effect to experimental animals and / or contacting them with cells. Is analyzed.
第二の工程では、 実験動物及び Z又は細胞から生体関連物質を抽出する。 生体 関連物質とは、 DNA、 RNA, タンパク質、 ペプチド、 ペプチド核酸、 脂質、 抗 体等である。 それらの抽出方法は当業者により適宜選択される。  In the second step, biological substances are extracted from laboratory animals and Z or cells. Biologically related substances are DNA, RNA, protein, peptide, peptide nucleic acid, lipid, antibody and the like. Those extraction methods are appropriately selected by those skilled in the art.
第三の工程では、 抽出した生体関連物質又はその調製物を上述のプローブ又は マイクロアレイに接触させる。 本発明の方法は、 上記接触後にプローブ又はその 調製物から発せられたシグナルを検出し、 そのシグナルの検出結果に基づいて被 験物質のアレルゲン性及び/又は抗アレルギー性を判定する工程を含む。 「調製 物」 とは、 例えば抽出した生体関連物質を RI、 蛍光等で標識したもの、 PCR、 T7 増幅法を使用して増幅した核酸類、 断片化した核酸等である。 生体関連物質 又はその調製物を遺伝子又はマイクロアレイに接触させることにより、 プローブ との反応性を観察することができる。 In the third step, the extracted biological substance or its preparation is brought into contact with the above-described probe or microarray. The method of the present invention provides a probe or its probe after the contact. A step of detecting a signal emitted from the preparation and determining the allergenicity and / or antiallergenicity of the test substance based on the detection result of the signal. “Preparations” are, for example, extracted biological substances labeled with RI, fluorescence, etc., nucleic acids amplified using PCR, T7 amplification, fragmented nucleic acids, and the like. The reactivity with the probe can be observed by bringing the biological substance or preparation thereof into contact with the gene or microarray.
反応条件は使用する生体関連物質の種類、 プローブの種類等により適宜選択さ れる。 反応終了後、 適当な検出手段により、 生体関連物質とプローブとの反応性 を評価する。 例えば生体関連物質が蛍光標識されている場合は、 蛍光顕微鏡によ り蛍光強度を測定することによりァレルゲン性及び/又は抗ァレルギ一性の評 価を行う。 ここで「評価」 とは、例えば測定した蛍光強度から発現量を定量化し、 対照区と比較することにより被験物質に暴露した際に発現している特定の遺伝 子の種類、 及びその発現量を特定することである。  Reaction conditions are appropriately selected depending on the type of biological substance to be used, the type of probe, and the like. After completion of the reaction, the reactivity between the biological substance and the probe is evaluated by an appropriate detection means. For example, when a biological substance is fluorescently labeled, the allergenicity and / or antiallergenicity is evaluated by measuring the fluorescence intensity with a fluorescence microscope. Here, “evaluation” means, for example, quantifying the expression level from the measured fluorescence intensity and comparing it with the control group to determine the type of the specific gene expressed when exposed to the test substance, and its expression level. It is to identify.
本発明のプローブは、 I型アレルギー又は IV型アレルギーに対するアレルゲン 性及び/又は抗ァレルギ一性判定に使用することができる。 本発明のプローブ又はマイクロアレイを使用することによって、 簡便なアレル ゲン性の判定及び検查をすることが可能となるため、 アレルゲン性のある食品に 適正表示をすることが容易となる。 これは、 健全な食生活の構築、 アレルギー予 防、 食品企業への信頼感の構築に役立ち、 産業の進展に結びつく。  The probe of the present invention can be used to determine allergenicity and / or antiallergenicity against type I allergy or type IV allergy. By using the probe or microarray of the present invention, it is possible to easily determine and examine allergenicity, so that it is easy to properly display foods with allergenicity. This helps to build a healthy diet, prevent allergies, and build confidence in food companies, leading to industrial progress.
また、 本発明によって簡便に抗アレルギー性の判定及び検査をすることが可能 となるため、 医薬品、 食品の研究開発現場で候補物質のスクリーニングに用いる ことができる。 従って、 本発明によって、 新規医薬品、 新規機能性食品開発の効 率化又は迅速化、 開発リスクの低減、 開発費用の削減ができる点で、 本発明のプ ローブ、 マイクロアレイ及び方法は極めて有用である。  In addition, since the present invention makes it possible to easily determine and test antiallergic properties, it can be used for screening candidate substances at the research and development sites for pharmaceuticals and foods. Therefore, the probe, microarray and method of the present invention are extremely useful in that the present invention can increase the efficiency or speed of the development of new pharmaceuticals and new functional foods, reduce development risks, and reduce development costs. .
さらに、 本発明は、 前記プローブ又はマイクロアレイを含む、 アレルゲン性及 びノ又は抗アレルギー性判定用キッ卜を提供する。  Furthermore, the present invention provides an allergenic and non-allergic anti-allergic kit comprising the probe or microarray.
本発明のキットは、上記マイクロアレイ以外に、他の成分を含んでいてもよい。 他の成分としては、例えば、 蛍光試薬、 制限酵素、 各種緩衝液、滅菌水、洗浄剤、 界面活性剤、 実験操作マニュアル （説明書） 等が挙げられる。 The kit of the present invention may contain other components in addition to the microarray. Examples of other components include fluorescent reagents, restriction enzymes, various buffers, sterilized water, detergents, surfactants, and experimental operation manuals (instructions).
以下、 実施例により本発明をさらに具体的に説明する。 但し、 本発明はこれら 実施例により限定されるものではない。  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
〔実施例 1〕 Example 1
マイクロアレイの製造  Microarray manufacturing
1 . 配置プローブの調製  1. Placement probe preparation
まず、 実験に使用するマイクロアレイとして貫通孔型のマイクロアレイを選び、 D N Aマイクロアレイに配置するためのプローブ核酸分子として、 表 1に記載し た配列情報をもつ 5'末端ビニル化核酸分子 （65mer) を用いた。  First, a through-hole type microarray was selected as the microarray to be used in the experiment, and a 5'-terminal vinylated nucleic acid molecule (65mer) having the sequence information shown in Table 1 was used as the probe nucleic acid molecule for placement on the DNA microarray. It was.
表 1 . において No.104 は IL-1ひ遺伝子に対応するプローブであり、 No.105 が IL- 1 ^6遺伝子、 No.107がIL·2遺伝子、No. l03が IL- 18遺伝子、 No.19が CCL2 遺伝子、 No.24が CCL3遺伝子、 No.25が CCL4遺伝子、 No.26が CCL 5遺伝 子及び No.208が TNF 遺伝子に対応するプローブである。 In Table 1, No. 104 is a probe corresponding to IL-1 gene, No. 105 is IL-1 ^ 6 gene, No. 107 is IL-2 gene, No. 103 is IL-18 gene, No. .19 is the CCL2 gene, No.24 is the CCL3 gene, No.25 is the CCL4 gene, No.26 is the CCL5 gene, and No.208 is the probe corresponding to the TNF gene.
表 1 table 1
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Figure imgf000018_0001
Figure imgf000019_0001
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68C.0 / .00Zdf / X3d £ 690 / 800Z OAV
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C.0/.00Jdf/X3d ひ £690/800Z OAV 〔実施例 2〕
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C.0 / .00 Jdf / X3d £ £ 690 / 800Z OAV Example 2
1 . 検体試料調製  1. Sample preparation
本実施例においては、 アレルギー様の状態を惹起できる物質として LPS (Lipopolysaccharide) を用い、 アレルギー様の状態に対し抑制効果のある物質 として TPCK (Modified ： 1J- (tosylamido - 2 - phenyl) ethyl chloro methyl ketone を用い、 細胞として: RAW246.7細胞 （マウスマクロファージ由来培養細胞） を用 いた。  In this example, LPS (Lipopolysaccharide) is used as a substance capable of causing an allergic state, and TPCK (Modified: 1J- (tosylamido-2-phenyl) ethyl chloromethyl) is used as a substance having an inhibitory effect on the allergic state. Using ketone, RAW246.7 cells (cultured cells derived from mouse macrophages) were used as cells.
RAW246.7細胞を LPS刺激単独、及び LPS刺激且つ TPCKlOOpmol存在下で 6時間培養し、 それぞれ細胞を回収し、 それぞれ Total RNAを抽出した。 Total RNAの抽出は RNeasy MinElute Kit (QIAGEN社製）を用いて実施した。 LPS 刺激の単独刺激群はァレルギ一惹起モデル群、 TPCK共存下での培養群がァレル ギー抑制モデル群とした。  RAW246.7 cells were cultured for 6 hours in the presence of LPS stimulation alone and in the presence of LPS stimulation and TPCKlOOpmol, and the cells were collected and total RNA was extracted. Total RNA was extracted using RNeasy MinElute Kit (QIAGEN). The single stimulation group for LPS stimulation was the allergy induction model group, and the culture group in the presence of TPCK was the allergy suppression model group.
それぞれの群の Total RNAを用いて、本発明のマイクロアレイにハイブリダイ ゼーシヨンさせる Biotin標識 aRNAを調製し、 ハイブリダィゼ一シヨンに供し た。 Biotin標識 aRNAの調製は Message Amp II Biotin Kit (Ambion社製)を用 いて、 所定の方法に従って実施した。  Using each group of total RNA, Biotin-labeled aRNA to be hybridized to the microarray of the present invention was prepared and subjected to hybridization. Biotin-labeled aRNA was prepared according to a predetermined method using Message Amp II Biotin Kit (Ambion).
2. マイクロアレイよるアツセィ  2. Atssay by microarray
ハイプリダイゼーシヨンバッファー溶液を以下のように調製した。  A hybridization solution was prepared as follows.
(0.12M TNT溶液： 1000ml)  (0.12M TNT solution: 1000ml)
1M Tris-HCl 120ml  1M Tris-HCl 120ml
1M NaCl 120ml  1M NaCl 120ml
0.5%Tween20 100ml  0.5% Tween20 100ml
蒸留水で 1000m 1にメスアップ  Measure up to 1000m 1 with distilled water
Biotin標識 aRNAを、 ハイブリダイゼ一シヨンバッファ一溶液 （0.12M TNT 溶液）に 15 g Z lSO 1 に懸濁させ、 三菱レイヨン社製マイクロアレイ (GenoPa ) を使用して 65 で 1 7時間ハイブリダイゼーション反応を行つ た。 反応後チップは、 洗浄バッファ一溶液 （0.12M TNT溶液） を用いて 65°Cで 2 0分間の洗浄を 2回行い、 最後に 2 x SSCで 10分間洗浄した。 チップの検出 は三菱レイョン社製検出器を用いて行った。 Biotin-labeled aRNA is suspended in 15 g Z lSO 1 in a hybridization buffer solution (0.12M TNT solution), and a hybridization reaction is carried out at 65 for 17 hours using a microarray (GenoPa) manufactured by Mitsubishi Rayon. The After the reaction, the chip is washed at 65 ° C using a washing buffer solution (0.12M TNT solution). Two 20 minute washes were performed, and a final wash with 2 x SSC for 10 minutes. Chip detection was performed using a detector manufactured by Mitsubishi Rayon.
蛍光標識は、 あ らかじめ蒸留水によって lmg Z ml に溶解した Cy5-Streptavdin(GE Healthcare社製)を 1/500に蒸留水で希釈した溶液 5mlに、 マイクロアレイを 30分間浸漬させることで行った。 浸漬後のマイクロアレイは 5mlの 0.12M TNT溶液によって 5分間、 4回洗浄に供した後、 三菱レイヨン製 DNAチップ検出器により、 蛍光強度を測定した。  Fluorescent labeling was performed by immersing the microarray for 30 minutes in 5 ml of a 1: 500 dilution of Cy5-Streptavdin (GE Healthcare) dissolved in lmg Z ml with distilled water. . After immersion, the microarray was washed 4 times for 5 minutes with 5 ml of 0.12M TNT solution, and the fluorescence intensity was measured with a DNA chip detector manufactured by Mitsubishi Rayon.
3. DNAチップよる解析結果 3. Results of DNA chip analysis
測定した蛍光強度は、 各群 GAPD遺伝子 （NM_008084) の蛍光強度を基準と して補正を行い、 各群における対象遺伝子の蛍光強度を比較した。  The measured fluorescence intensity was corrected based on the fluorescence intensity of each group GAPD gene (NM_008084), and the fluorescence intensity of the target gene in each group was compared.
その結果、 GAPD遺伝子の（LPS単独刺激群の蛍光強度値/ TPCK刺激群の蛍 光強度値） は（1 4 7 1 5 7 / 1 4 5 9 5 3 )であり、 比は 1.01であった。そのため GAPDの相対的発現差を log (底は 2 )で表した場合 0となり、その他の遺伝子の結 果について、 相対的発現差を求めた （図 1 )。  As a result, the GAPD gene (LPS single stimulation group fluorescence intensity value / TPCK stimulation group fluorescence intensity value) was (1 4 7 1 5 7/1 4 5 9 5 3), and the ratio was 1.01. . Therefore, when the relative expression difference of GAPD was expressed in log (bottom 2), it was 0, and the relative expression difference was calculated for the results of other genes (Fig. 1).
図 1より、 LPS刺激によって亢進した遺伝子群である Blnk, Camk2d, Caspll, CCL2, CCL5, F52, Gbpl, IL- lOra, IL- 18, IL-1 , IL- 1 β， Lyn, Trafl, Trim30が TPCK刺激によって抑制されていることが示された。  From Fig. 1, Blnk, Camk2d, Caspll, CCL2, CCL5, F52, Gbpl, IL-lOra, IL-18, IL-1, IL-1 β, Lyn, Trafl, Trim30, which are enhanced by LPS stimulation It was shown to be suppressed by TPCK stimulation.
〔実施例 3〕 Example 3
本実施例においては、 アレルギー様の状態を惹起できる物質として LPS(Lipopolysaccharide：グラム陰性菌由来リポ多糖類)及び OVA (卵白アルブミ ン： ovalbumin)を用いた。 なお、 LPSは炎症反応を惹起させる、 いわば IV型ァ レルギ一に特徴的な反応を誘起することができる。 また OVAを経口投与するこ とで食物アレルギーのアレルゲンとなり、 I 型アレルギーに特徴的な反応を誘起 することができる。  In this example, LPS (Lipopolysaccharide: Gram-negative bacterium-derived lipopolysaccharide) and OVA (egg white albumin: ovalbumin) were used as substances capable of inducing an allergic state. LPS induces an inflammatory response, that is, a response characteristic of type IV allergy. Oral administration of OVA becomes an allergen for food allergies and can induce a characteristic response to type I allergies.
それら物質を、 以下の条件でマウスに投与し、 脾臓を採取し、 脾臓細胞中の mRNAをマイクロアレイで測定した。 結果は図 2及び図 3に示した。  These substances were administered to mice under the following conditions, the spleen was collected, and mRNA in the spleen cells was measured with a microarray. The results are shown in FIGS.
<実験条件 > •使用マウス C 57 BL 早 8週令 <Experimental conditions> • Use mouse C 57 BL early 8 weeks old
[LPS]  [LPS]
'経口投与条件： 1回投与、 0日のみ、 l OmgZmlを 0. 2mlマウスに経 口投与。  'Oral administration conditions: 1 dose, 0 days only, l OmgZml administered orally to 0.2 ml mice.
4時間後に脾臓を摘出。  Four hours later, the spleen was removed.
COVA)  COVA)
'経口投与条件： 2回投与、 0日、 8日目、 10mg/mlを 0. 2mlマウ スに経口投与。  'Oral administration conditions: 2 doses, day 0, day 8, 10 mg / ml administered orally to 0.2 ml mice.
• 21日後に脾臓を摘出。  • The spleen was removed after 21 days.
〔RNAの抽出及び標識化〕  [RNA extraction and labeling]
•実施例 2と同様に処理を行った。  • Treated as in Example 2.
' マイクロアレイよるアツセィ及び DNAチップよる解析結果は、 実施例 2と同 様に行った。 'The analysis results using the microarray and the DNA chip were the same as in Example 2.
その結果、 LPS刺激で顕著に発現するのは IL-la.IL-l|S, CCL2, CCL3, CCL4, CCL5, TNFo!であることが分かった。 また OVA投与時には IL-2, IL-18が充進 することが分かった （図 2)。  As a result, it was found that IL-la.IL-l | S, CCL2, CCL3, CCL4, CCL5, TNFo! In addition, IL-2 and IL-18 were found to be enhanced when OVA was administered (Fig. 2).
即ち、 IV型アレルギーの場合は （IL-lQ!，IL-li6，TNFQi,CCL2等） が亢進し、 I型アレルギーの場合は IL-2、 IL-18が亢進することが示唆され、 これらの遺伝 子発現を観察することにより、 食物アレルギーの型の判定を行うことが可能とな つた。  In other words, in the case of type IV allergy (IL-lQ !, IL-li6, TNFQi, CCL2, etc.) is enhanced, and in case of type I allergy, it is suggested that IL-2 and IL-18 are enhanced. By observing gene expression, it became possible to determine the type of food allergy.
〔実施例 4〕 Example 4
本実施例においては、 アレルギー様の状態を惹起できる物質として LPS (Lipopolysaccharide ： グ ラ ム 陰性菌 由 来 リ ポ多糖類）及び PMA (12-O-Tetradecanoylp orbol 13-acetate)を用いた。 なお、 LPS は炎症反応を惹 起させる、 いわば] V型アレルギーに特徴的な反応を誘起することができる。 また PMA は各種サイト力イン分泌を促進し、 IV型アレルギーとは異なる反応を誘起 する。 それら物質を、 以下の条件でマウス細胞に添加して刺激した。 これらの細胞群 より Total UNAを抽出し、各遺伝子の mRNA発現をマイクロアレイで測定した。 結果は図 4及び図 5に示した。 In this example, LPS (Lipopolysaccharide: lipopolysaccharide derived from gram-negative bacteria) and PMA (12-O-tetradecanoylp orbol 13-acetate) were used as substances capable of inducing an allergic state. LPS induces an inflammatory response, in other words, it can induce a characteristic response to type V allergy. PMA also promotes secretion of various site forces and induces a reaction different from type IV allergy. These substances were added to mouse cells and stimulated under the following conditions. Total UNA was extracted from these cell groups, and mRNA expression of each gene was measured with a microarray. The results are shown in FIG. 4 and FIG.
'使用細胞 RAW 2 6 4 . 7細胞 （マウスマクロファージ様細胞）  'Used cells RAW 2 6 4 .7 cells (mouse macrophage-like cells)
ぐ実験条件 > Experimental conditions>
〔LPS〕  [LPS]
• 1 X 1 0 6 cells/ml の細胞に LPS を最終濃度 1 0 g /m 1で添加して刺 激、 6時間後細胞を回収して RNAを抽出 • 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]  CPMA]
· 1 X 1 0 6 cells/ml の細胞に PMAを最終濃度 1 g /m 1で添加して刺激、 6時間後細胞を回収して RNAを抽出 · Stimulate by adding PMA to 1 X 10 6 cells / ml at a final concentration of 1 g / m 1, 6 hours later, collect cells and extract RNA
〔RNAの抽出及び標識化〕  [RNA extraction and labeling]
•実施例 2と同様に処理を行った。 . 様に行った。  • Treated as in Example 2. I went to
その結果、 LPS刺激では図 4の遺伝子群は顕著に発現が亢進している力 PMA 刺激では IL-l Q!、 IL-2，、 CCL2はそれほど上昇が認められない。  As a result, the expression of the gene group in Fig. 4 is remarkably increased by LPS stimulation. IL-l Q !, IL-2, and CCL2 are not significantly increased by PMA stimulation.
この結果より IL-la、 IL-2、 CCL2 を観察することはアレルギーのタイプ判別 に有効であると考えられる。 産業上の利用可能性  From these results, it is considered that observing IL-la, IL-2, and CCL2 is effective in determining the type of allergy. Industrial applicability
本発明により、 食品成分によるアレルギー発症又は抑制作用の有無を効率的か つ効果的に判定をすることができるオリゴヌクレオチドプローブ及びマイクロ アレイが提供される。 また、 本発明のオリゴヌクレオチドプローブ及びマイクロ アレイは、 未知の食品成分のアレルゲン性を判定又は検査したり、 機能性食品成 分の開発段階におけるアレルゲン性又は抗アレルギー性作用を判定するための 簡便なツールとしても使用することができる。 配列表フリーテキスト 配列番号 1〜 2 2 1 ：合成 DNA According to the present invention, there are provided an oligonucleotide probe and a microarray that can efficiently and effectively determine the presence or absence of an allergy onset or a suppressive action due to food components. In addition, the oligonucleotide probe and microarray of the present invention are easy to determine or test the allergenicity of unknown food ingredients, or to determine allergenic or antiallergic effects in the development stage of functional food ingredients. It can also be used as a tool. Sequence Listing Free Text SEQ ID NOs: 1-2 2 1: Synthetic DNA

Claims

請求 の 範 囲 The scope of the claims
1 . 食物アレルギー応答に関連する遺伝子の塩基配列のうち、 少なくとも一部の 塩基配列にハイプリダイズすることができる塩基配列を含む、 ァレルゲン性 及び Z又は抗ァレルギ一性判定用オリゴヌクレオチドプローブ。 1. An oligonucleotide probe for determining allergenicity and Z or antiallergenicity, comprising a nucleotide sequence that can be hybridized to at least a portion of the nucleotide sequence of a gene related to food allergy response.
2 . 食物アレルギー応答に関連する遺伝子が、 IL-l o!遺伝子、 IL-l iS遺伝子、 IL-2 遺伝子、 ]X-18遺伝子、 CCL2遺伝子、 CC 3遺伝子、 CCL4遺伝子、 CCL 5 遺伝子及び TNF a遺伝子からなる群から選ばれる少なくとも 1つである、 請 求項 1記載のオリゴヌクレオチドプロ一ブ。  2. The genes related to food allergic response are IL-lo! Gene, IL-l iS gene, IL-2 gene,] X-18 gene, CCL2 gene, CC 3 gene, CCL4 gene, CCL 5 gene and TNF The oligonucleotide probe according to claim 1, which is at least one selected from the group consisting of a genes.
3 . アレルゲン性及び/又は抗アレルギー性判定が、 I型アレルギー又は IV型ァ レルギ一に対するものである請求項 1又は 2記載のオリゴヌクレオチドプロ ーブ。 3. The oligonucleotide probe according to claim 1 or 2, wherein the allergenicity and / or antiallergic determination is for type I allergy or type IV allergy.
4 . 請求項 1〜 3のいずれかに記載のオリゴヌクレオチドプローブが基盤に配置 された、 ァレルゲン性及び Z又は抗ァレルギ一性判定用マイクロアレイ。  4. A microarray for determining allergenicity and Z or antiallergenicity, wherein the oligonucleotide probe according to any one of claims 1 to 3 is arranged on a substrate.
5 . 複数の貫通孔を有し、 それら貫通孔にゲルが保持されているマイクロアレイ であって、 前記ゲルに請求項 1〜 3のいずれかに記載のオリゴヌクレオチド プローブが保持されている、 アレルゲン性及び Z又は抗アレルギー性判定用 マイクロアレイ。 5. A microarray having a plurality of through-holes and a gel held in the through-holes, wherein the oligonucleotide probe according to any one of claims 1 to 3 is held in the gel. And Z or anti-allergic microarray.
6 . オリゴヌクレオチドプローブが 1 0 0 0種類以下である、 請求項 4又は 5に 記載のマイクロアレイ。  6. The microarray according to claim 4 or 5, wherein the number of oligonucleotide probes is 100 or less.
7 . 以下の工程を含む、 被験物質のァレルゲン性及び/又は抗ァレルギ一性の判 定方法：  7. Method for determining the allergenicity and / or antiallergenicity of a test substance, including the following steps:
( 1 ) 被験物質を実験動物に投与し若しくは接触させ、 及び 又は被験物質 を細胞に接触させる工程、  (1) administering or contacting a test substance to a laboratory animal, and / or contacting a test substance with a cell;
( 2 ) 実験動物及び/又は細胞から生体関連物質を抽出する工程、 並びに (2) a process for extracting biological substances from laboratory animals and / or cells, and
( 3 ) 前記生体関連物質又はその調製物を、請求項 1〜 3のいずれかに記載の オリゴヌクレオチドプローブ又は請求項 4〜 6のいずれかに記載のマイク口 アレイに接触させる工程。 (3) A step of bringing the biological substance or a preparation thereof into contact with the oligonucleotide probe according to any one of claims 1 to 3 or the microphone mouth array according to any one of claims 4 to 6.
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