JP5965569B2 - Allergenic or anti-allergic probe - Google Patents

Description

  The present invention relates to oligonucleotide probes used for determining the allergenicity and / or allergenicity of food components and a microarray on which they are arranged.

In recent years, the risk of food allergies has been increasing along with changes in dietary habits, such as the introduction of crops with no dietary experience into foods and increased consumption of specific foods. At present, it is estimated that about 1 to 3% of Japanese are food allergic patients (National Life Center). In addition, consumer needs for food safety and reliability have been increasing year by year, and in particular regarding the inclusion of allergens, it is obliged to label raw materials of processed foods for specific substances. Thus, the current situation is that the relationship between allergens in foods and safety is becoming more serious, and it is necessary to develop a simple and reproducible determination method of allergenicity for unknown allergens.
In addition, with the increase in allergic patients, ingredients contained in foods are required to have highly safe allergy suppression or alleviation action, scientifically elucidating the allergenic action of food, It is also important to aim to develop foods that do not cause allergies.
Conventionally, the ELISA method has been used as a method for determining allergenicity. However, this method only detects a known allergenic substance, and cannot detect an allergen for an unknown substance. For this reason, there is no method to determine the allergenicity of an unknown crop that has never been eaten, except by estimating it by long-term administration to animals, and a simple method for determining allergenicity has been known so far. It is not done. In addition, when determining the allergenicity and antiallergic action of food components, it is necessary to consider the combined effect of various foods, that is, the effect that allergenicity changes when multiple foods are used together. It is practically difficult to perform the above-mentioned by the ELISA method, and it is considered that such a change can be confirmed only by analyzing the expression behavior of a plurality of genes.
In recent years, research on biological genome information such as human genome decoding has progressed remarkably, and post-genomic research has been actively conducted to apply this information to actual diagnosis or treatment activities, especially in the medical field. . 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 very small area. By detecting the presence or amount of gene fragments in a sample by a hybridization reaction between nucleic acids, a plurality of gene fragments are detected. It is possible to easily analyze gene expression behavior.
Currently, DNA microarrays manufactured by photolithography and spotting are mainly used as research tools. However, as a platform unique to the present applicant that applies fiber and resin technology, “fiber type DNA microarrays” (Trade name Genopal) "(references 1 to 3) has been developed and marketed (URL: http://www.mrc.co.jp/genome/). This fiber-type DNA microarray is characterized in that a high-precision DNA microarray can be mass-produced at low cost by employing an original manufacturing method. Further, since operations in the array manufacturing process do not require skill, there is no variation in test results due to individual differences among test workers, and further, there is no need to consider individual differences for each experimental animal. Therefore, accurate data can be easily obtained by using the fiber DNA microarray.
By the way, as a research related to the onset and suppression of allergies, food ingredients that show inflammatory cytokine production suppression effects and degranulation suppression effects related to allergy suppression effects have been clarified and the detailed mechanism of action has been studied so far. And elucidation is underway (Reference 4). As a result, basic knowledge about genes related to the onset and suppression of allergies has been obtained.
In recent years, genetically modified foods and substances that have never been used in foods have many unknown allergenic and anti-allergenic properties. However, the present condition is that many of these foods are distributed in the market. Therefore, knowing the allergenicity and antiallergic properties of foods is extremely important for those who have an allergic constitution. Whether or not these have allergenicity and the like is determined by observing allergic symptoms by continuously administering foods of unknown allergenicity to experimental animals over a period of several months or longer. However, even with this time-consuming study, the results obtained did not exceed speculation.
References 1. Japanese Patent No. 3515470. Patent No. 3510882 Specification 2. Japanese Patent No. 35155704. Kobori et al. Cell Death Differ. , 11 (1) 123-130 (2004)

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 to provide a DNA microarray in which they are arranged.
As a result of earnest research to solve the above problems, the present inventor has reproducibility of allergens or antiallergenicity of food ingredients by combining knowledge about the onset or suppression of allergies caused by food ingredients and DNA microarray technology. An efficient, simple and highly reproducible gene group was found. In addition, the present inventors have succeeded in developing a DNA microarray in which they are arranged, thereby completing the present invention.
That is, the present invention is an allergenic and / or antiallergic oligonucleotide probe comprising a nucleotide sequence capable of hybridizing to at least a part of the nucleotide sequence of a gene related to food allergy response. is there.
The gene associated with food allergy response is selected from the group consisting of IL-1α gene, IL-1β gene, IL-2 gene, IL-18 gene, CCL2 gene, CCL3 gene, CCL4 gene, CCL5 gene and TNFα gene, for example At least one of them. In the present invention, the allergenicity and / or antiallergic determination is, for example, for type I allergy or type IV allergy.
Furthermore, the present invention is a microarray for determining allergenicity and / or antiallergenicity, on which the above-described oligonucleotide probe is arranged.
Furthermore, the present invention is a microarray having a plurality of through-holes, in which a gel is held in the through-holes, wherein the oligonucleotide probe is held in the gel, allergenic and / or anti-allergic properties It is a microarray for determination.
In the microarray of the present invention, the number of oligonucleotide probes is preferably 1000 or less.
Furthermore, the present invention is a method for determining the allergenicity and / or antiallergenicity of a test substance, comprising the following steps:
(1) administering or contacting a test substance to a laboratory animal and / or contacting the test substance with cells;
(2) A step of extracting a biological substance from a laboratory animal and / or a cell, and (3) a step of bringing the biological substance or a preparation thereof into contact with the oligonucleotide probe or the microarray.
Embodiments of the present invention will be described below. The following embodiments are merely examples for explaining the present invention, and the present invention is not intended to be limited only to these embodiments. The present invention can be implemented in various forms without departing from the gist thereof.
It should be noted that all publications cited in the present specification, for example, prior art documents and 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.
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 used as a simple tool for determining or examining the allergenicity of unknown food ingredients, or for determining allergenic or antiallergic effects in the development stage of functional food ingredients. Can also be used.
In the present invention, the method for determining the allergenicity and / or antiallergenicity of a food component comprises extracting a nucleic acid from a laboratory animal or cultured cell to which an allergic unknown food or the like has been administered in a short period of 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 in which they are arranged. According to the method of the present invention, the evaluation of allergic properties and the like can be shortened, labor-saving, and the results can be stabilized, and the combined effects of several kinds of foods can be determined.

FIG. 1 is a diagram showing a gene group whose expression is suppressed by TPCK.
FIG. 2 is a diagram showing the results of determining the state of allergy induction using LPS and OVA.
FIG. 3 is a diagram showing the results of determining the state of allergy induction using LPS and OVA.
FIG. 4 is a diagram showing the results of determining the state of allergy induction using LPS and PMA.
FIG. 5 is a diagram showing the results of determining the state of allergy induction using LPS and PMA.

The present invention has been completed by eating foods containing allergens or food candidate substances that are supposed to contain allergens, or by paying attention to genes that change when these foods are contacted. The present invention relates to a microarray in which oligonucleotide probes having a base sequence that partially hybridizes are arranged. That is, the present invention is a microarray in which oligonucleotide probes related to a plurality of food allergy responses are arranged.
“Food allergic response” refers to an abnormal immune response caused by an excessive immune response caused by an orally ingested or nasally inhaled food component or a food component in contact with the skin. It refers to a disordered reaction that induces Food allergies include (a) allergen-specific IgE antibodies that cause immediate symptoms, or (b) allergen-specific inflammatory T cells, eosinophils or macrophages that cause delayed swelling or tissue destruction. There is something to wake up. Food allergies are likely 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.), potatoes, seafood (abalone, squid, , Shrimp, crab, salmon, mackerel, etc.), fruits (orange, kiwi fruit, peach, apple, fig, etc.).
Examples of allergens include ovalbumin and ovomucoid in eggs, casein and β-lactoglobulin in milk, gliadin and α-amylase inhibitor in wheat, 2S albumin in peanuts, and parvalbumin in fish and shellfish. And tropomyosin.
In the present invention, the “oligonucleotide probe” refers to a probe that serves as a probe for detecting a gene whose expression level changes in association with the promotion or suppression of an allergic response. As such a probe, for example, when an allergic substance is produced when an immune response occurs due to ingestion of foods with allergens, the expression level changes when allergens are produced, or expressed when inhibitory substances are produced to suppress allergic reactions Examples include oligonucleotides for detecting genes whose amount changes (hereinafter referred to as probes).
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 an allergic response, but is a gene group related to various cytokines, histamine, etc. Is preferred. In particular, it is a gene group of the minimum unit useful for discriminating type I allergy and type IV allergy that are typical as food allergies.
Here, “type I allergy” is an allergy that reacts immediately after contact with an antigen (allergen). IgE class antibodies bind to Fcε receptors on mast cells at their Fc portion. When an antigen binds to this, the Fc receptors bind to each other through the antigen molecule. As a result, histamine is released from mast cells to the outside of the cell, and chemical mediators such as leukotriene and platelet activating factor are synthesized and released. Chemical mediators cause increased vascular permeability, smooth muscle contraction, leukocyte chemotaxis, etc., and as a result, symptoms appear due to tissue reaction.
“Type IV allergy” refers to tissue injury caused by a reaction between an antigen and a T cell without antibody involvement. T cells produce lymphokines such as interleukin (IL), which lead to macrophage and neutrophil infiltration, plasma exudation due to increased vascular permeability, fibroblast proliferation, and the like.
In the present invention, such a group of genes related to allergy is the subject. These gene groups include IL-1 (interleukin) α gene, IL-1 β gene, IL-2 gene, IL-4 gene, IL-8 gene, IL-10 gene, IL-18 gene, CCL2 (chemokine) ) Gene, CCL3 gene, CCL4 gene, CCL5 gene, PTGS2 (prostaglandin-endoperoxide synthase 2) and TNF (tumor necrosis factor) α gene, more preferably IL-1α gene, IL-1β gene, These are IL-2 gene, IL-18 gene, CCL2 gene, CCL3 gene, CCL4 gene, CCL5 gene and TNFα gene. These genes can be used alone or in combination. Preferably, the behavior of all these genes is observed and used for determination.
The oligonucleotide used as a probe in the present invention is capable of hybridizing with at least a part of the base sequences of these genes. 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 hybridized 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 having a similar base sequence in DNA encoding these proteins or peptides. Therefore, depending on the base sequence of the selected probe, the sequences may be similar to each other. With such a base sequence, a plurality of genes cannot be distinguished and detected. 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. In designing the probe, in addition to selecting a specific region, the distance from the 3 terminal is within 1500 bases, Tm (melting temperature) is 60 to 80 ° C., GC in the probe It is preferable to design in consideration of the base content of 40 to 60% and the ease of forming the secondary structure. These conditions are shown in Table 1 described later.
In order to design the base sequence, homology between the base sequences may be searched using an existing program between the genes from which the probes to be arranged are derived, and a portion having low homology may be selected. 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 designed probe length is 10 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).
The probe is capable of hybridizing with the detection target gene when hybridization with the sample is performed as described above. Therefore, it is necessary to consider stringency in hybridization when designing a probe. By making stringency close to some extent, even if similar base sequence regions exist between genes, other different regions can be distinguished and hybridized. In addition, when the base sequences between genes are almost different, stringency can be set gently.
Such stringency conditions include, for example, hybridization under conditions of 65 ° C. to 68 ° C. under tight conditions, and hybridization under conditions of 37 ° C. to 55 ° C. under mild conditions. is there.
In the hybridization conditions of the present invention, stringent conditions include, for example, “0.12M Tris · HCl / 0.12M NaCl / 0.05% Tween-20, 50 ° C.”, “0.12M Tris · HCl / 0.12M NaCl / 0.05% Tween-20, 42 ° C. ”,“ 0.12M Tris · HCl / 0.12M NaCl / 0.05% Tween-20, 37 ° C. ”, more stringent conditions include For example, “0.12M Tris · HCl / 0.12M NaCl / 0.05% Tween-20, 65 ° C.”, “0.12M Tris · HCl / 0.12M NaCl / 0.05% Tween-20, 68 ° C.” , "0.06M Tris.HCl / 0.06M NaCl / 0.05% Tween-20, 65 Mention may be made of the conditions ", and the like.
More specifically, the probe is added and kept at 65 ° C. for 1 hour or longer for hybridization, and then 20 minutes at 65 ° C. in 0.12 M Tris.HCl / 0.12 M NaCl / 0.05% Tween-20. There is also a method in which washing is performed 4 times, and finally, 0.12M Tris.HCl / 0.12M NaCl at 65 ° C. for 10 minutes. 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 the buffer salt concentration and temperature.
For the detailed procedure of the hybridization method, “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989), “Current Protocols in Molecular Biology” (John 7) You can refer to it.
In the present invention, the base sequence of the target gene for detection does not have to be the base sequence itself of each gene described above, and a part of the base sequence may be mutated by deletion, substitution, insertion, or the like. . Therefore, the target gene for detection hybridizes under stringent conditions with a sequence complementary to the base sequence represented by the SEQ ID No. described in the above table, 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. 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 probes are 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 a light-induced solid layer on a silicon base A method (photolithographic method) in which probes are sequentially synthesized by reaction is known.
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. 2000-78998).
(1) Drill a resin block so that a parallel hole is formed with a laser or the like.
(2) A gel precursor solution containing a probe is introduced into the hole, and a gelation reaction is performed in the hole.
(3) Repeat the cutting of the resin block in the direction of transverse cutting of the hole.
Moreover, as a preferable example of a through-hole array, a hollow fiber type microarray can be illustrated (refer Unexamined-Japanese-Patent No. 2000-245461, Unexamined-Japanese-Patent No. 2001-239594, and Unexamined-Japanese-Patent No. 2003-344400). 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) The fibers are aligned by passing the hollow fibers through holes of a plurality of jigs having holes having the same pattern as the target arrangement pattern.
(2) After expanding the interval of the jig to a desired width, the fiber bundle is fixed by filling a matrix resin between the fibers. Thereby, a block (array) in which the fiber bundle is fixed by the resin can be obtained.
(3) A gel precursor solution containing a probe is introduced into the hollow portion of each fiber, a gelation reaction is performed in the hollow portion, and the probe is fixed to the gel.
(4) Cut in a direction crossing the longitudinal direction of the fiber.
This cutting means slicing the block in a direction intersecting with the longitudinal direction, whereby a slice of the array can be obtained. A probe is fixed to the fiber portion of the thin piece.
The manufacturing method of the said fiber type microarray is described in Unexamined-Japanese-Patent No. 2001-239594, A fiber type microarray can be manufactured by referring to the said gazette. Further, it is preferable that a gel such as acrylamide is held in the hollow portion, and the oligonucleotide (probe) is fixed (held) on the gel. Such fixation allows the probes to be arranged three-dimensionally within the gel and maintain a three-dimensional structure. Therefore, the detection efficiency is increased compared to a planar microarray in which a probe is bonded to a slide glass whose surface is coated, and it is possible to perform a highly sensitive and highly reproducible inspection.
Further, the number of types of probes arranged in the microarray is preferably 1000 or less, preferably 500 or less, and more preferably 250 or less 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, if they have different base sequences, they are specified as different types.
Next, the test method for the allergenicity and / or antiallergic property of the test substance will be described.
The present invention is an allergenicity and / or antiallergic test method for a test substance, comprising the following steps.
(1) administering or contacting a test substance to a laboratory animal and / or contacting a test substance with cells;
(2) A step of extracting a biological substance from a laboratory animal and / or a cell, and (3) a step of bringing the biological substance or a preparation thereof into contact with the above-mentioned oligonucleotide or microarray.
“Allergenic” refers to the property of a food that functions as an allergen when consumed, 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-recognizing T cells to induce antigen-specific T cells and antibody production. The type of cells or antibodies to be induced and the symptoms of allergic reaction vary depending on the condition of the sensitized living body (for example, genetic factor, nutritional state) or the allergen invasion route.
“Anti-allergic” means the property of suppressing an allergic response. Since this allergic response includes many steps, there is a possibility that allergy can be suppressed in each step. Therefore, the substance having anti-allergic properties may function at any step of the development of allergy as long as the allergic response is suppressed. Examples of the food containing an antiallergic component include tea and the like, and examples of the antiallergic component include green tea catechin.
In the first step, a test substance is administered to or contacted with a laboratory animal and / or brought into contact with a cell. The “test substance” is a substance to be examined. Examples thereof include food, a solution obtained by homogenizing the same, and a crude product thereof. The form in which the test substance (test food) is administered to or contacted with the experimental animal is not limited, and any of oral administration and parenteral administration (injection, transdermal route, etc.) may be used. Moreover, the aspect made to contact with a cell means both adding a candidate substance in a cell culture solution and culturing a cell with a candidate substance, for example. The test substance may be administered to the experimental animal for a short period of several days. When cells are used, they are not limited to the cell cycle period, and any phase of cells may be used.
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 beverages are used as test substances, they can be used in the form of liquids or diluted in an appropriate buffer, and when solids are used as test substances, they can be homogenized in an appropriate buffer or culture medium. Thereafter, it can be diluted to a concentration suitable for culture or administration.
Examples of the cell type 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 / or contacted with a cell. By this step, for example, the expression of all or part of the IL-1α gene, IL-1β gene, IL-2 gene, IL-18 gene, CCL2 gene and TNFα gene is enhanced, but subsequently, the allergy suppressing effect It is analyzed whether or not enhancement is suppressed in all or a part of the above-mentioned genes by administering or contacting a certain test substance to an experimental animal and / or contacting a cell.
In the second step, a biological substance is extracted from the experimental animal and / or cell. The biological substance includes DNA, RNA, protein, peptide, peptide nucleic acid, lipid, antibody and the like. Those extraction methods are appropriately selected by those skilled in the art.
In the third step, the extracted biological material or preparation thereof is brought into contact with the above-described probe or microarray. The method of the present invention includes a step of detecting a signal emitted from the probe or its preparation after the contact and determining the allergenicity and / or antiallergic property of the test substance based on the detection result of the signal. The “preparation” is, for example, an extracted biological material labeled with RI, fluorescence or the like, a nucleic acid amplified using PCR or T7 amplification method, a fragmented nucleic acid, or the like. The reactivity with a probe can be observed by bringing a biological substance or a preparation thereof into contact with a gene or a microarray.
The 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 the 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 specify the type of the specific gene expressed when exposed to the test substance and the expression level. It is to be.
The probe of the present invention can be used for allergenicity and / or antiallergic determination for type I allergy or type IV allergy.
By using the probe or microarray of the present invention, it is possible to easily determine and test 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.
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 development of new pharmaceuticals and new functional foods, reduce development risks, and reduce development costs.
Furthermore, the present invention provides an allergenic and / or antiallergic determination kit comprising the probe or microarray.
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 buffer solutions, sterilized water, cleaning agents, surfactants, and experimental operation manuals (instructions).
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Manufacture of microarrays Preparation of Arrangement Probe First, a through-hole type microarray was selected as a 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 a probe nucleic acid molecule for arrangement on the DNA microarray. ) Was used.
Table 1. No. 104 is a probe corresponding to the IL-1α gene. 105 is the IL-1β gene, no. 107 is the IL-2 gene, No. 103 is the 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 a probe corresponding to the TNFα gene.

1. Specimen sample preparation In this example, LPS (Lipopolysaccharide) is used as a substance capable of causing an allergic state, and TPCK (Modified: L- (tosylamido-2-phenyl)) is used as a substance having an inhibitory effect on the allergic state. Ethyl chloromethyl ketone) and 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, LPS stimulation and 100 pmol of TPCK, the cells were collected, and total RNA was extracted from each. Total RNA was extracted using RNeasy MinElute Kit (manufactured by QIAGEN). The single stimulation group of LPS stimulation was an allergy-inducing model group, and the culture group in the presence of TPCK was an allergy suppression model group.
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. Microarray assay A hybridization buffer solution was prepared as follows.
(0.12M TNT solution: 1000 ml)
1M Tris-HCl 120ml
120 ml of 1M NaCl
0.5% Tween20 100ml
Make up to 1000 ml with distilled water. Biotin-labeled aRNA was suspended in a hybridization buffer solution (0.12 M TNT solution) at 15 μg / 150 μl, and then used for 17 hours at 65 ° C. using a microarray (GenoPal ^™ ) manufactured by Mitsubishi Rayon. A hybridization reaction was performed. After the reaction, the chip was washed twice at 65 ° C. for 20 minutes using a washing buffer solution (0.12M TNT solution), and finally washed with 2 × SSC for 10 minutes. The chip was detected using a detector manufactured by Mitsubishi Rayon.
The fluorescent labeling was performed by immersing the microarray in 5 ml of a solution obtained by diluting Cy5-Streptavdin (GE Healthcare) dissolved in distilled water in advance to 1/500 with distilled water for 30 minutes. After the immersion, the microarray was washed 4 times with 5 ml of 0.12M TNT solution for 5 minutes, and the fluorescence intensity was measured with a DNA chip detector manufactured by Mitsubishi Rayon.
3. Results of analysis by DNA chip The measured fluorescence intensity was corrected with reference to the fluorescence intensity of each group GAPD gene (NM_008084), and the fluorescence intensity of the target gene in each group was compared.
As a result, the GAPD gene (LPS single stimulation group fluorescence intensity value / TPCK stimulation group fluorescence intensity value) was (147157/145959), and the ratio was 1.01. Therefore, when the relative expression difference of GAPD was expressed by log (bottom 2), it was 0, and the relative expression difference was obtained for the results of other genes (FIG. 1).
From FIG. 1, Blk, Camk2d, Casp11, CCL2, CCL5, F52, Gbp1, IL-10ra, IL-18, IL-1α, IL-1β, Lyn, Traf1, and Trim30, which are genes enhanced by LPS stimulation, are TPCK. It was shown to be suppressed by stimulation.

In this example, LPS (Lipopolysaccharide: Gram-negative bacterium-derived lipopolysaccharide) and OVA (ovalbumin) were used as substances that can induce an allergic state. LPS can induce an inflammatory reaction, that is, a reaction characteristic of type IV allergy. Oral administration of OVA becomes an allergen for food allergies, and can induce a reaction characteristic of type I allergy.
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 FIG. 2 and FIG.
<Experimental conditions>
・ Mouse C57BL ♀ 8 weeks old [LPS]
-Oral administration conditions: 1 administration, 0 day only, 10 mg / ml orally administered to 0.2 ml mice.
Four hours later, the spleen was removed.
[OVA]
-Oral administration conditions: 2 doses, 0 days, 8 days, 10 mg / ml orally administered to 0.2 ml mice.
・ After 21 days, the spleen was removed.
[RNA extraction and labeling]
The treatment was performed in the same manner as in Example 2.
The assay using the microarray and the analysis result using the DNA chip were performed in the same manner as in Example 2.
As a result, IL-1α. It was found to be IL-1β, CCL2, CCL3, CCL4, CCL5, TNFα. It was also found that IL-2 and IL-18 were enhanced when OVA was administered (FIG. 2).
That is, in the case of type IV allergy (IL-1α, IL-1β, TNFα, CCL2, etc.) is enhanced, and in the case of type I allergy, it is suggested that IL-2 and IL-18 are enhanced. By observing the expression, it was possible to determine the type of food allergy.

In this example, LPS (Lipopolysaccharide: Gram-negative bacterium-derived lipopolysaccharide) and PMA (12-O-tetradecanoylphorbol 13-acetate) were used as substances capable of inducing an allergic state. LPS can induce an inflammatory reaction, that is, a reaction characteristic of type IV allergy. PMA also promotes secretion of various cytokines and induces a reaction different from type IV allergy.
These substances were added to mouse cells and stimulated under the following conditions. Total RNA was extracted from these cell groups, and mRNA expression of each gene was measured with a microarray. The results are shown in FIGS.
・ Used cells RAW264.7 cells (mouse macrophage-like cells)
<Experimental conditions>
[LPS]
・ Stimulate by adding LPS to the cell of 1 × 10 ⁶ cells / ml at a final concentration of 10 μg / ml, and after 6 hours, collect the cells and extract RNA [PMA]
・ Stimulate by adding PMA to 1 × 10 ⁶ cells / ml at a final concentration of 1 μg / ml, collect cells 6 hours later and extract RNA [RNA extraction and labeling]
The treatment was performed in the same manner as in Example 2.
The assay using the microarray and the analysis result using the DNA chip were performed in the same manner as in Example 2.
As a result, the expression of the gene group in FIG. 4 is remarkably increased by LPS stimulation, but IL-1α, IL-2, and CCL2 are not significantly increased by PMA stimulation.
From these results, it is considered that observing IL-1a, IL-2, and CCL2 is effective for allergic type discrimination.

  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 used as a simple tool for determining or examining the allergenicity of unknown food ingredients, or for determining allergenic or antiallergic effects in the development stage of functional food ingredients. Can also be used.

SEQ ID NOs: 1-21: Synthetic DNA
[Sequence Listing]

Claims (5)

Oligos for determining allergenicity and / or antiallergenicity using RAW264.7 cells, including a nucleotide sequence capable of hybridizing to at least a part of the nucleotide sequences of genes related to food allergy response A nucleotide probe comprising:
Genes associated with food allergy response, Blnk, Camk2d, Casp11, CCL2 , CCL5, F52, Gbp1, IL-10ra, IL-18, Lyn, Ri Oh at least one gene selected from the group consisting of Traf1 and Trim30 ,
The allergenicity is a property of raising the same state as allergic-like state induced by LPS, the anti-allergic properties, Ru properties der having the same effect as suppressing effect on the state of allergy-like by TPCK,
The oligonucleotide probe. A microarray for determining allergenicity and / or antiallergic property, wherein the oligonucleotide probe according to claim 1 is arranged on a substrate ,
The allergenicity is a property that causes the same state as an allergic state caused by LPS, and the antiallergic property is a property that has the same effect as an inhibitory effect on an allergic state caused by TPCK.
The microarray . A microarray having a plurality of through-holes and a gel held in the through-holes, wherein the oligonucleotide probe according to claim 1 is held in the gel. a use microarray,
The allergenicity is a property that causes the same state as an allergic state caused by LPS, and the antiallergic property is a property that has the same effect as an inhibitory effect on an allergic state caused by TPCK.
The microarray .   The microarray according to claim 2 or 3, wherein the number of oligonucleotide probes is 1000 or less. As the following this:
(1) A step of bringing a test substance into contact with RAW264.7 cells,
(2) a step of extracting total RNA from the cells; and (3) the oligonucleotide probe according to claim 1 or the microarray according to any one of claims 2 to 4, wherein the total RNA or a preparation thereof is used. The process of contacting
A method for determining allergenicity and / or antiallergenicity of a test substance, comprising:
The allergenicity is a property that causes the same state as an allergic state caused by LPS, and the antiallergic property is a property that has the same effect as an inhibitory effect on an allergic state caused by TPCK.
The determination method .

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