A method for constructing an animal model of hazelnut food allergy

Abstract

The invention relates to a method for constructing an animal model of hazelnut food allergy, and specifically to an animal model of food allergy constructed by using an allergen Cor a 14. Compared with the existing hazelnut protein food allergy animal model, the animal model has the advantages of a single allergen, obvious sensitization effect, etc., and can be applied to experimental research on hazelnut food allergy, providing a research model for clinical research on hazelnut allergy or drug screening against hazelnut allergy.

Description

Technical Field

[0001] The invention relates to a method for constructing an animal model of hazelnut food allergy, in particular to an animal model of food allergy constructed by using an allergen Cor a 14, and belongs to the field of food biotechnology. Background Art

[0002] Food allergy, also known as food hypersensitivity, refers to the body's abnormal immune response to food allergens (usually proteins) that enter the human body, which usually leads to physiological dysfunction or tissue damage, and triggers a series of clinical symptoms, such as urticaria on the skin, respiratory asthma, cough, gastrointestinal nausea, vomiting, diarrhea, etc. In severe cases, shock or even death. As a major public health and food safety issue of global concern, the incidence of food allergies has increased at a rate of 10% per year, and has shown a trend of diversification, complexity and widening, seriously affecting the health of people with allergies.

[0003] Nuts are one of the main types of foods rich in allergens, among which peanuts and hazelnuts are typical. Hazelnut allergy usually starts in childhood and adolescence, and basically does not disappear with age. Mild hazelnut allergy manifests as oral itching, while severe hazelnut allergy can be life-threatening. According to the literature reported so far, hazelnut protein contains a variety of allergen components, such as Cor a 1, Cor a 2, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, Cor a 13, Cor a 14 and Cor a TLP, etc., and the allergen components are very complex. Therefore, when the body has a hazelnut allergic reaction, it is impossible to determine which specific allergen affects the body.

[0004] In order to accurately study the specific mechanism of allergic reactions caused by hazelnut allergens and find more effective treatment methods, it is necessary to construct an animal model of allergy to a single hazelnut allergen to provide a theoretical basis for the precise clinical diagnosis of hazelnut allergy.

[0005] The specific selection of hazelnut allergen components and how to construct an effective allergic animal model for experimental research on hazelnut food allergy to provide a research model for clinical research or drug screening of hazelnut allergy are all urgent issues to be solved by technical personnel in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for constructing an animal model of hazelnut food allergy, wherein the construction method comprises using the allergen Cor a 14 to perform sensitization treatment and stimulation treatment on non-human mammals to construct the animal model of hazelnut food allergy.

[0007] In a preferred embodiment of the present invention, the non-human mammal is a mouse. More preferably, the mouse is of the BALB / c strain.

[0008] In a preferred embodiment of the present invention, the sensitization treatment and the challenge treatment are carried out by oral gavage and / or intraperitoneal injection.

[0009] In a preferred embodiment of the present invention, the non-human mammal is a BALB / c mouse; during the sensitization treatment stage, the dosage of the allergen Cor a 14 is 30 to 100 mg / kg body weight each time.

[0010] In a preferred embodiment of the present invention, during the sensitization treatment stage, the dosage of the allergen Cor a 14 is 50 mg / kg body weight each time.

[0011] In a preferred embodiment of the present invention, the sensitization treatment and the stimulation treatment are carried out by oral gavage; during the sensitization treatment stage, cholera toxin CT is used as a mucosal immune adjuvant, and the dosage of cholera toxin CT is 0.3-1 mg / kg body weight each time; and gavage sensitization is performed twice a week for four consecutive weeks; the stimulation treatment is carried out on the fourth day after the end of the sensitization treatment stage.

[0012] In a preferred embodiment of the present invention, the allergen Cor a 14 is prepared by the following steps:

[0013] Step 1): using n-hexane to defatted hazelnut powder to prepare defatted hazelnut protein powder;

[0014] Step 2): extracting the defatted hazelnut protein powder with a glycine-hydrochloric acid buffer to obtain the allergen Cor a 14.

[0015] In another aspect, the present invention provides the use of the animal model constructed by the above construction method in screening drugs against hazelnut food allergy or in experimental research on hazelnut food allergy.

[0016] In another aspect, the present invention provides a method for screening potential substances against hazelnut allergy, the method comprising: applying the candidate substance to the animal model constructed by the above construction method, and detecting the allergic reaction index of the animal model.

[0017] Compared with the existing hazelnut protein food allergy animal model, the food allergy animal model using the allergen Cor a 14 of the present invention has the advantages of a single allergen and obvious sensitization effect, and can be applied to experimental research on hazelnut food allergy, and provides a research model for clinical research on hazelnut allergy or drug screening against hazelnut allergy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The statistical results of the allergic clinical symptom scores of mice in each group;

[0019] Figure 2 The statistical results of the changes in rectal temperature of each group of mice before and after intragastric gavage challenge;

[0020] Figure 3 The results of the detection of allergen-specific IgE content in the serum of each group of mice after intragastric gavage challenge;

[0021] Figure 4 The results are the test results of serum histamine content in each group of mice after intragastric administration;

[0022] Figure 5 This is the SDS-PAGE gel electrophoresis result of the target protein separated and purified in Example 1. DETAILED DESCRIPTION

[0023] After extensive and in-depth research, the inventors of the present application unexpectedly found that the animal model constructed using the allergen Cor a 14 can well simulate the food allergy reaction of hazelnuts. Compared with the existing hazelnut protein food allergy animal model, the food allergy animal model using the allergen Cor a 14 of the present application has the advantages of a single allergen and obvious sensitization effect, and is more suitable for experimental research on hazelnut food allergy, providing a research model for clinical research on hazelnut allergy or drug screening against hazelnut allergy.

[0024] The "hazelnut food allergy", also known as "hazelnut protein allergy", refers to the abnormal immune response produced by mammals after eating hazelnut food / hazelnut protein, which is caused by allergens and usually leads to disorders of the body's physiological functions or tissue damage, and triggers a series of clinical symptoms. The main clinical symptoms are some or all of the following: urticaria on the skin, respiratory asthma, cough, gastrointestinal nausea, vomiting, diarrhea, etc., and in severe cases, shock or even death; the main clinical detection characteristics are some or all of the following: increased IgE levels in serum, increased inflammatory cells in the respiratory tract or lungs, increased eosinophils in the lungs, and increased levels of histamine and inflammatory cytokines in serum.

[0025] Hazelnut protein generally refers to the defatted protein powder separated from hazelnut kernels. The protein content is usually around 40%. It is rich in 8 amino acids needed by the human body and is a common type of allergenic protein in nut foods.

[0026] The "hazelnut protein" that causes hazelnut food allergy can be naturally present, for example, it can be a protein isolated and purified from natural hazelnut kernels; it can also be artificially prepared, for example, recombinant hazelnut protein can be produced according to conventional genetic recombination technology.

[0027] The "hazelnut protein" that triggers hazelnut food allergy may also be a fragment, derivative or analog of hazelnut protein. The terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity of natural hazelnut protein, for example, a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, a polypeptide having a substitution group in one or more amino acid residues, or a polypeptide formed by the fusion of an additional amino acid sequence to this sequence.

[0028] The "hazelnut protein" that causes hazelnut food allergy may also be a modified or improved hazelnut protein, for example, a hazelnut protein that has been modified or improved to improve its half-life, effectiveness, metabolism, and / or protein potency. Any changes that do not affect the biological activity of the hazelnut protein are included.

[0029] Regarding the selection of animals for constructing animal models: In this application, "the non-human mammals" include but are not limited to: mice, rats, rabbits, dogs, monkeys, etc. The non-human mammals are relatively close to humans in terms of genome composition, organ anatomy, individual development, metabolic mode and disease pathogenesis, and can be well applied to the research on the pathogenesis of human diseases and drug pharmacology.

[0030] The hazelnut food allergy animal model of the invention is prepared by immunizing non-human mammals with the allergen Cora 14 separated and purified from hazelnut protein.

[0031] Preferably, in a specific embodiment of the present application, the non-human mammal is a mouse. More preferably, in an example of the present application, the mouse strain used is BALB / c.

[0032] Regarding the immune process, it includes sensitization and stimulation.

[0033] In a specific embodiment of the present application, the sensitization treatment and the stimulation treatment are carried out by oral gavage and / or intraperitoneal injection; that is, oral gavage can be used alone, or intraperitoneal injection can be used alone, or a combination of oral gavage and intraperitoneal injection can be used.

[0034] In a preferred embodiment of the present invention, the sensitization treatment and the challenge treatment are separately carried out by oral gavage to perform systemic immunization of non-human mammals with hazelnut allergen Cor a 14. Based on the basic theory in this field, oral gavage or intraperitoneal injection are both conventional systemic immunization methods. The present application provides an animal model constructed by oral gavage. Based on this scheme, those skilled in the art can use intraperitoneal injection to replace or partially replace it.

[0035] In a preferred embodiment of the present invention, the non-human mammal is a BALB / c mouse; during the sensitization treatment stage, the dosage of the allergen Cor a 14 is 30 to 100 mg / kg body weight each time. The dosage of the allergen Cor a 14 can be 30, 40, 50, 60, 70, 80, 90 or 100 mg / kg body weight each time. More preferably, the dosage of the allergen Cor a 14 is 50 mg / kg body weight.

[0036] In a specific embodiment of the present invention, the sensitization treatment is performed by oral gavage; during the sensitization treatment, the allergen and the mucosal immune adjuvant are co-immunized to complete the induction of the animal model. The mucosal immune adjuvant is an immune adjuvant commonly used in the oral gavage method, which is used to assist immune induction.

[0037] In a preferred embodiment of the present invention, the sensitization treatment and the stimulation treatment are carried out by oral gavage; during the sensitization treatment stage, cholera toxin CT is used as a mucosal immune adjuvant, and the dosage of cholera toxin CT is 0.3-1 mg / kg body weight each time; and gavage sensitization is performed twice a week for four consecutive weeks; the stimulation treatment is carried out on the fourth day after the end of the sensitization treatment stage.

[0038] After completing the sensitization induction and stimulation of animals with hazelnut protein allergen Cor a 14, the inventors of the present application used a variety of allergic reaction indicators to verify the success of the model, such as "allergic clinical symptom score", "rectal temperature", "serum allergen-specific IgE content" and "serum histamine content". It was verified that after sensitization induction and stimulation with hazelnut protein allergen Cor a 14, mice showed obvious clinical symptoms of allergies, rectal temperature decreased significantly, and allergen-specific IgE levels and histamine content in mouse serum increased significantly, and there was no significant difference with the relevant data of hazelnut protein. Combining these indicators, it can be seen that the inventors of the present application successfully constructed a mouse model of hazelnut food allergy using the allergen Cor a 14.

[0039] The hazelnut food allergy animal model constructed by the present invention can be used for the study of the pathogenesis of hazelnut food allergy, such as the intestinal mucosal immune system, sensitization-related intestinal flora and marked differential metabolites, and the mechanism at the cellular level or molecular level. The mechanism of allergy (immune response) caused by hazelnut food is studied and explained, providing a way to develop drugs for treating hazelnut food allergy.

[0040] The hazelnut food allergy animal model constructed by the present invention can be used to screen drugs against hazelnut allergy.

[0041] The present invention provides a method for screening potential substances for resisting hazelnut allergy, the method comprising: applying a candidate substance to an animal model of hazelnut food allergy constructed by the method of the present invention, detecting the allergic clinical symptoms, rectal temperature, serum allergen-specific IgE content or serum histamine content of the animal model; if the allergic clinical symptoms are alleviated, the rectal temperature rises, the serum allergen-specific IgE content decreases, and the serum histamine content decreases, it indicates that the candidate substance is a potential substance for resisting hazelnut allergy.

[0042] Preferably, a control group is also set up, which is an animal model in which the candidate substance is not administered and other conditions are the same as those of the experimental group to which the candidate substance is administered; by comparison, if the relevant allergic reaction indicators of the experimental group, such as the allergen-specific IgE content in the serum or the histamine content in the serum, are statistically significantly lower than the data of the control group, it indicates that the candidate substance is a potential substance for resisting hazelnut allergy.

[0043] The potential substances screened out above can constitute a screening library, and these potential substances can be further subjected to cell tests, animal tests and / or clinical trials to further confirm the effects of these potential substances.

[0044] The present invention is further described below by way of examples, but the present invention is not limited to these specific embodiments.

[0045] Example 1: Isolation and purification of allergen Cor a 14 from hazelnut powder;

[0046] The separation and purification of the allergen Cor a 14 from hazelnut powder involves the following two steps:

[0047] Step 1): using n-hexane to defatted hazelnut powder to prepare defatted hazelnut protein powder;

[0048] Specifically in this embodiment, hazelnut cake (purchased from Liaoyang Dihao Agriculture Co., Ltd.) was first obtained, crushed and sieved through an 80-mesh sieve, and 20 g of hazelnut powder with a particle size of less than 80 mesh was taken; 400 mL of n-hexane was added to the obtained 20 g of hazelnut powder, stirred at about 25° C. for 4 h, and centrifuged at 4° C. and 8000 rpm / min for 20 min, and the lower layer of precipitate was taken, dried and sieved through an 80-mesh sieve to obtain defatted hazelnut protein powder;

[0049] Step 2): extracting the defatted hazelnut protein powder with a glycine-hydrochloric acid buffer;

[0050] 5 g of defatted hazelnut protein powder obtained in step 1) was added to 40 mL of glycine-hydrochloric acid buffer, stirred at 37° C. for 1 h, and then centrifuged at 4° C. and 8000 rpm / min for 20 min to obtain the supernatant; the supernatant was further purified by dialysis to obtain a PBS solution of the target protein.

[0051] Product identification: The PBS solution of the target protein obtained by separation and purification in step 2) above was subjected to SDS-PAGE gel electrophoresis. The electrophoresis pattern results are shown in Figure 5 The target protein band was cut out for protein spectrum identification; according to the mass spectrometry identification results and comparison with the Uniprot database, it was determined that the target protein obtained by separation and purification was hazelnut allergen Cor a 14.

[0052] The protein content of the PBS solution of the target protein obtained above was determined by the BCA method (Bicinchoninic Acid Assay), and the protein concentration was adjusted and placed in a -80 degree Celsius refrigerator for future use.

[0053] Example 2: Construction of a mouse model of food allergy to hazelnut allergen

[0054] 1. Experimental Animals

[0055] The experimental animals used in this application are BALB / c mice, which were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and were raised in the animal room of China Agricultural University (sterile free diet, breeding environment temperature 22-25 degrees Celsius, humidity 40-60%) and related experimental operations.

[0056] After one week of adaptive feeding, the following construction experiment was conducted on BALB / c mice. The animals were randomly divided into 4 groups according to their body weight, with two subgroups in each group. They were allowed to drink water and eat freely and were weighed weekly.

[0057] 2. Build the experiment

[0058] The experimental process of this embodiment is divided into "allergen sensitization treatment stage" and "allergen provocation treatment".

[0059] Allergen sensitization treatment stage:

[0060] The mice were sensitized by gavage twice a week for four consecutive weeks, and the gavage dose during this stage was 300 μL / mice.

[0061] Experimental group: The intragastric administration solution was 300 μL of a buffer solution (PBS buffer) containing allergen Cor a 14 (50 mg / kg mouse body weight) and CT (0.5 mg / kg mouse body weight); wherein the allergen Cor a 14 was obtained by separation and purification in the above Example 1; and CT (0.5 mg / kg mouse body weight) was a conventional mucosal immune adjuvant cholera toxin.

[0062] Blank control group 1: intragastric administration solution was 300 μL PBS buffer;

[0063] Blank control group 2: intragastrically administered solution was 300 μL of PBS buffer containing CT (0.5 mg / kg mouse body weight);

[0064] Positive control group 1: intragastrically administered solution was 300 μL of PBS buffer solution containing hazelnut protein (50 mg / kg mouse body weight) (defatted hazelnut protein powder obtained in step 1 of the above Example 1, the same below);

[0065] Positive control group 2: The intragastric administration solution was 300 μL of PBS buffer containing hazelnut protein (250 mg / kg mouse body weight).

[0066] Allergen challenge treatment:

[0067] On the 4th day after the allergen sensitization stage, the mice were gavaged and challenged with a dose of 500 μL / mouse.

[0068] Experimental group: The intragastric administration solution was 500 μL of PBS buffer containing the allergen Cor a 14 (500 mg / kg mouse body weight) (the intragastric administration solution did not contain CT, and the content of the allergen Cor a 14 was 10 times that of the sensitization stage);

[0069] Blank control group 1: intragastric administration solution was 500 μL PBS buffer;

[0070] Blank control group 2: intragastric administration solution was 500 μL PBS buffer;

[0071] Positive control group 1: The intragastric administration solution was 500 μL of PBS buffer containing hazelnut protein (500 mg / kg mouse body weight).

[0072] Positive control group 2: The intragastric administration solution was 500 μL of PBS buffer containing hazelnut protein (2500 mg / kg mouse body weight).

[0073] 3. Detection of allergic reaction indicators

[0074] In the examples of the present application, the detected allergic reaction indicators include "allergic clinical symptom score", "rectal temperature", "serum allergen-specific IgE content" and "serum histamine content".

[0075] 1) Allergy clinical symptom score

[0076] 30 minutes after the above-mentioned allergen challenge treatment (i.e. 30 minutes after oral gavage challenge), the mice in each group were scored for clinical allergy symptoms. The scoring criteria are shown in Table 1 below.

[0077] Table 1

[0078]

[0079] The statistical results of allergy clinical symptom scores can be found in Figure 1 .from Figure 1 It can be seen that the allergic clinical symptom score of mice in the experimental group (2.50±1.05) was significantly higher than that of the blank control group 1 (0.00) and the blank control group 2 (0.33±0.52); there was no significant difference between the positive control group 1 (2.83±0.75) and the positive control group 2 (2.67±0.82).

[0080] This application Figure 1 The results were analyzed using ANOVA statistical method. Figure 2-4 The results were also obtained using the same statistical analysis method.

[0081] Therefore, judging from the "allergy clinical symptom score" indicator, the mouse model constructed in this application using the allergen Cor a 14 can simulate the mouse model of hazelnut protein food allergy.

[0082] 2) Rectal temperature measurement

[0083] 30 minutes before and 30 minutes after the above-mentioned allergen stimulation treatment "gastric stimulation", a rectal thermometer probe coated with vaseline was inserted into the rectum of each group of mice, and the readings were taken after the temperature stabilized. Repeat three times to calculate the average value.

[0084] The results of the rectal temperature changes of each group of mice before and after gavage stimulation are shown in Figure 2 .from Figure 2 It can be seen that the rectal temperature change value of the mice in the experimental group (-1.88±0.42℃) was significantly lower than that of the blank control group 1 (0.80±0.35), blank control group 2 (0.18±0.51), positive control group 1 (0.00±0.55℃) and positive control group 2 (-0.12±0.54℃).

[0085] From the comparison results of rectal temperature changes in each group, the mouse model constructed by the allergen Cor a 14 in the present application has a significantly lower rectal temperature drop value than the result of the mouse model of hazelnut protein food allergy, which shows that the mouse model constructed by the allergen Cor a 14 in the present application has a more obvious sensitizing effect and higher sensitivity.

[0086] 3) Detection of allergen-specific IgE content in serum

[0087] 24 hours after the above-mentioned allergen challenge treatment "gastric challenge", blood was collected from mice in each group (blood was collected by removing the eyeballs), and the level of allergen-specific IgE in the serum of mice in each group was measured.

[0088] The specific operation method of allergen-specific IgE content detection is as follows:

[0089] a) Coating: Add 100 μL of 1 mg / mL allergen solution (Cor a 14 in PBS) diluted with coating buffer to each well of a 96-well Costar plate and coat overnight at 4°C (12 h);

[0090] b) Washing: discard the coating solution, drain on absorbent paper, add 200 μL 0.05% PBST washing solution (PBS + 0.05% Tween 20) to each well with a spray gun, let it stand for 1 min each time and shake it slightly, then spin dry on absorbent paper, wash 4 times;

[0091] c) Blocking: Add 150 μL of 1% BSA / PBS blocking solution to each well and block at 37°C for 1 h;

[0092] d) Washing: discard the blocking solution, drain on absorbent paper, add 200 μL 0.05% PBST washing solution to each well with a dispenser, let stand for 1 min each time and shake gently, then spin dry on absorbent paper, wash 4 times;

[0093] e) Add serum: add 100 μL of serum diluted with 1% BSA / PBS serum diluent to each well and incubate at 37°C for 2 h;

[0094] f) Washing: discard the blocking solution, drain on absorbent paper, add 200 μL 0.05% PBST washing solution to each well with a dispenser, let stand for 1 min each time and shake slightly, then spin dry on absorbent paper, wash 4 times;

[0095] g) Add secondary antibody: add 100 μL of diluted enzyme-labeled secondary antibody HRP-IgE to each well and incubate at 37°C for 1 h;

[0096] h) Washing: discard the blocking solution, drain on absorbent paper, add 200 μL 0.05% PBST washing solution to each well with a spray gun, let stand for 1 min each time and shake slightly, then spin dry on absorbent paper, wash 6 times;

[0097] i) Add substrate: add 100 μL TMB to each well for color development and place at 37°C in the dark for 5-30 min;

[0098] j) Termination and measurement: Add 50 μL of stop solution to each well to terminate the color development reaction, and measure the OD value of each well at a wavelength of 450 nm using an enzyme reader.

[0099] The results of allergen-specific IgE content detection in each group are shown in Figure 3 .from Figure 3 It can be seen that the level of allergen-specific IgE in the serum of mice in the experimental group (OD 450 =0.11±0.01) was significantly higher than that of the blank control group 1 (OD 450 =0.07±0.00) and blank control group 2 (OD 450 =0.08±0.01); compared with the positive control group 1 (OD 450 =0.12±0.02) and positive control group 2 (OD 450 =0.12±0.01) had no significant difference.

[0100] Therefore, from the perspective of the "allergen-specific IgE content" indicator, the mouse model constructed in this application using the allergen Cor a14 can simulate the mouse model of hazelnut protein food allergy.

[0101] 4) Detection of histamine content in serum

[0102] 24 hours after the above-mentioned allergen challenge treatment "gastric challenge", blood was collected from mice in each group (blood was collected by removing the eyeballs), and the histamine content in the serum of mice in each group was measured.

[0103] This application uses an ELISA kit purchased from Wuhan Cloud-Clone Technology Co., Ltd. to detect the histamine content in the serum of each group of mice. The specific operation process is shown in the kit manual and will not be repeated in this article.

[0104] The results of histamine content in each group can be found in Figure 4 .from Figure 4 It can be seen that the histamine content in the serum of mice in the experimental group (3.43±0.61) was significantly higher than that in the blank control group 1 (2.38±0.26) and the blank control group 2 (1.72±0.13); there was no significant difference between the experimental group and the positive control group 1 (3.26±0.71) and the positive control group 2 (3.48±0.69).

[0105] Therefore, from the perspective of the "allergen-specific IgE content" indicator, the mouse model constructed in this application using the allergen Cor a14 can simulate the mouse model of hazelnut protein food allergy.

[0106] Based on the results of 1)-4) above, the mouse model constructed by sensitization and stimulation treatment with the allergen Cor a 14 in the present application showed that the results of various allergic reaction indicators were significantly higher than those of the two blank control groups, among which the results of three allergic reaction indicators were basically consistent with the results of the hazelnut protein allergic mouse model, and the performance in the decrease of rectal temperature was more sensitive than the hazelnut protein allergic mouse model.

[0107] It should be understood that although this specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation mode may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0108] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of hazelnut food allergy, characterized in that: The construction method comprises using the allergen Cor a 14 to perform sensitization treatment and provocation treatment on non-human mammals to construct the hazelnut food allergy animal model; The non-human mammal is a BALB / c mouse; The sensitization treatment and the challenge treatment are carried out by oral gavage; During the sensitization treatment stage, the dosage of the allergen Cor a 14 is 30-100 mg / kg body weight each time; During the sensitization treatment stage, cholera toxin CT is used as a mucosal immune adjuvant, and the dosage of cholera toxin CT is 0.3-1 mg / kg body weight each time; and oral sensitization is performed twice a week for four consecutive weeks; the stimulation treatment is performed on the fourth day after the end of the sensitization treatment stage.

2. The construction method according to claim 1, characterized in that: During the sensitization treatment stage, the allergen Cor a14 was administered at a dose of 50 mg / kg body weight each time.

3. The construction method according to any one of claims 1 to 2, characterized in that: The allergen Cor a 14 is prepared by the following steps: Step 1): Using n-hexane to defatted hazelnut powder to prepare defatted hazelnut protein powder; Step 2): The defatted hazelnut protein powder is extracted with a glycine-hydrochloric acid buffer to obtain the allergen Cor a 14.

4. Use of the animal model constructed by the construction method according to any one of claims 1 to 2 in screening drugs against hazelnut food allergy.

5. A method for screening potential substances against hazelnut allergy, characterized in that: The method comprises: applying the candidate substance to the animal model constructed by the construction method according to any one of claims 1 to 2, and detecting the allergic reaction index of the animal model.

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