WO2008069342A1 - Sonde de détermination de caractère allergène ou anti-allergène - Google Patents

Sonde de détermination de caractère allergène ou anti-allergène 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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English (en)
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/ja
Publication of WO2008069342A1 publication Critical patent/WO2008069342A1/fr

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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

Abstract

La présente invention concerne: un oligonucléotide pour la détermination de caractère allergène et/ou anti-allergène, comprenant une séquence oligonucléotidique capable d'hybridation avec au moins une partie d'une séquence nucléotidique pour un gène apparenté à la réponse allergique à un aliment; un microréseau d'ADN pour la détermination de caractère allergène et/ou anti-allergène, comprenant un oligonucléotide disposé sur un substrat; et un procédé pour la détermination de caractère allergène et/ou anti-allergène mettant en œuvre le microréseau.
PCT/JP2007/073890 2006-12-05 2007-12-05 Sonde de détermination de caractère allergène ou anti-allergène WO2008069342A1 (fr)

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