Construction method of animal model of insect protein food allergy

Abstract

The invention discloses a construction method of an animal model for insect protein food allergy. The constructed animal model can be used for screening potential substances for resisting insect protein allergy. The food allergy animal model constructed by adopting the insect protein is applied to research on sensitization of edible insects serving as a novel protein source, and a research model is provided for clinical research on insect protein allergy or processing methods and substance screening for resisting insect allergy.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for constructing an animal model of insect protein food allergy, in particular to an animal model of insect protein food allergy constructed using insect protein as an allergen, belonging to the field of food biotechnology. BACKGROUND

[0002] Food allergy is a disease in which the immune system mistakenly recognizes certain food proteins as harmful antigens, triggering abnormal immune responses, usually involving complex mechanisms mediated by IgE or non-IgE. After patients ingest the sensitizing food, symptoms can involve multiple systems, such as itching, erythema, and urticaria on the skin, nasal itching, wheezing, and cough in the respiratory tract, nausea, vomiting, abdominal pain, and diarrhea in the digestive tract, and in severe cases, even anaphylactic shock symptoms such as laryngeal edema and decreased blood pressure. In recent years, the incidence of food allergy has been on the rise globally. According to data from the World Allergy Organization, the incidence of food allergy in children in developed countries is about 5%-8%, and in adults, it is about 1%-2%. Common allergens include milk, eggs, peanuts, nuts, fish, shellfish, wheat, and soybeans, and it has become an important public health problem.

[0003] Insects are considered a potential source of alternative protein to meet the huge demand for protein in the future. Currently, insects such as ox flies, yellow mealworms, locusts, and black powder beetles, crickets, and yellow mealworms have been approved as food ingredients in different countries and regions. The potential allergenicity of insect proteins can cause severe allergic reactions in consumers. Current research on insect protein allergenicity has mainly focused on cross-reactivity between insects and crustaceans. Paramyosin has been identified as a cross-reactive allergen between shrimp and various insects, including black water flies, crickets, and yellow mealworms. Arginine kinase is another widely cross-reactive pan-allergen. Cross-reactivity of arginine kinase in black water flies and domestic crickets has been verified by dot blotting with sera from shrimp or mite-allergic patients. Arginine kinase from silkworm pupae is the only allergen from edible insects (Bomb m 1) that has been officially recognized as a food allergen and included in the IUIS allergen database. Glycoprotein (27 kDa), chitinase, and mucin are reported to be potential allergens in silkworm pupae. Research on insect protein allergenicity is still at the stage of identifying allergens using allergic patient sera and describing their characteristics.

[0004] In order to accurately study the specific mechanisms of allergic reactions caused by insect protein allergens, an animal model of insect protein food allergy needs to be constructed to provide a theoretical basis for the clinical precise diagnosis of insect protein allergy. How to construct an effective allergic animal model for experimental research on insect-containing food allergy and provide a research model for clinical research or allergenicity reduction processing methods of insect protein allergy are problems that need to be solved by those skilled in the art. SUMMARY

[0005] The present application aims at the existing problems, and provides a method for constructing an animal model of food allergy to insect protein.

[0006] The present application is implemented by the following technical scheme: a method for constructing an animal model of food allergy to insect protein, comprising the following contents:

[0007] S1, extracting insect protein from insects, adding the insect protein and cholera toxin into a buffer solution to obtain a sensitization solution, and adding the insect protein into a buffer solution to obtain an excitation solution, wherein the content of the insect protein in the excitation solution is 4-6 times the content of the insect protein in the sensitization solution;

[0008] S2, performing a sensitization treatment on mice by gavage using the sensitization solution, and continuously performing the sensitization treatment for 3-5 weeks;

[0009] S3, performing an excitation treatment on the mice by gavage using the excitation solution at 6-8 days after the sensitization treatment;

[0010] S4, performing an allergic clinical symptom score on the mice after the excitation treatment, and determining the sensitization of the insect protein according to the allergic clinical symptom score of the mice.

[0011] In an embodiment of the present application, the buffer solution is a PBS buffer.

[0012] In an embodiment of the present application, the mice are of the BALB / c strain.

[0013] In an embodiment of the present application, the dose of the insect protein for administration during the sensitization treatment is 30-80 mg / kg of body weight each time. More preferably, the dose of the insect protein for administration during the sensitization treatment is 50 mg / kg of body weight each time.

[0014] In an embodiment of the present application, the dose of the cholera toxin for administration during the sensitization treatment is 8-12 μg per mouse each time. More preferably, the dose of the cholera toxin for administration during the sensitization treatment is 10 μg per mouse each time.

[0015] In an embodiment of the present application, the dose of the insect protein for administration during the excitation treatment is 150-400 mg / kg of body weight each time. More preferably, the dose of the insect protein for administration during the excitation treatment is 250 mg / kg of body weight each time.

[0016] In an embodiment of the present application, the extraction step of the insect protein is as follows:

[0017] S01. The dried insect bodies are washed with distilled water and 90% to 100% alcohol, and then dried and crushed to obtain insect powder;

[0018] S02, defatting the insect powder with petroleum ether and n-hexane to prepare defatted insect protein powder;

[0019] S03. Immerse the defatted insect protein powder in distilled water with a pH of 9 to 11 to extract the insect protein.

[0020] In one embodiment of the present invention, the sensitization treatment comprises gavage of the mice with the sensitization solution 1 to 3 times per week.

[0021] The second object of the present invention is to provide an animal model constructed by the construction method.

[0022] The third object of the present invention is to provide the application of the animal model in screening drugs against insect protein allergies.

[0023] Beneficial effects of the present invention:

[0024] The animal model constructed by this invention can effectively simulate the symptoms of insect protein food allergy and can be used to screen for potential anti-insect protein allergy substances. The invention uses insect protein to construct an animal model for food allergy, and its application in studying the allergenicity of edible insects as a novel protein source provides a research model for clinical research on insect protein allergy and for screening processing methods and substances to combat insect allergies. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Statistical results of weight changes of mice in each group during the sensitization stage;

[0026] Figure 2 Statistical results of allergic clinical symptom scores of mice in each group;

[0027] Figure 3 The results of serum IgE content detection in each group of mice after oral gavage challenge;

[0028] Figure 4 The results are the test results of histamine content in the serum of each group of mice after oral gavage challenge;

[0029] Figure 5 This is the SDS-PAGE gel electrophoresis result of the target protein separated and purified in Example 1. DETAILED DESCRIPTION

[0030] (1) The present application inventors have found through extensive and in-depth research that an allergenic animal model constructed using an allergen insect protein can well simulate insect food allergy reactions. The food allergy animal model of the present application using an allergen insect protein fills the gap in insect protein allergy mouse models and is applied to the study of the allergenicity of edible insects as a new source of protein, providing a research model for clinical research on insect protein allergy or the screening of processing methods and substances for anti-insect allergy.

[0031] (2) The "insect food allergy", also known as "insect protein allergy", refers to an abnormal immune response of a mammal after eating insect food / insect protein, which is caused by an allergen and usually leads to physiological dysfunction or tissue damage and triggers a series of clinical symptoms, with the main clinical symptoms being some or all of the following: hives on the skin, respiratory asthma, cough, gastrointestinal nausea, vomiting, diarrhea, and in severe cases, shock and even death; the main clinical detection features are some or all of the following: elevated serum IgE levels, increased respiratory or lung inflammatory cells, increased lung eosinophils, and increased serum histamine and inflammatory cytokine content.

[0032] (3) Insect protein generally refers to defatted protein powder isolated from insect bodies, with a protein content of more than 50% and rich in the eight amino acids required by the human body. It is a new type of allergenic protein discovered with the entry of insect protein into the food system.

[0033] (4) The "insect protein" that triggers insect food allergy can be naturally occurring, such as protein isolated and purified from insect bodies, or artificially prepared, such as recombinant insect protein produced according to conventional genetic recombination techniques.

[0034] (5) The "insect protein" that triggers insect protein food allergy can also be a fragment, derivative, or analog of insect protein. The terms "fragment", "derivative", and "analog" refer to polypeptides that substantially maintain the same biological function or activity of natural insect protein, for example, polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, polypeptides with substituent groups in one or more amino acid residues, or polypeptides formed by the fusion of additional amino acid sequences to this sequence.

[0035] (6) The "insect protein" that triggers insect food allergy can also be a modified or improved insect protein, such as an insect protein modified or improved to promote its half-life, effectiveness, metabolism, and / or protein efficacy. Any change that does not affect the biological activity of the insect protein is included.

[0036] (7) Selection of animals for constructing animal models: In the present application, "the non-human mammal" includes, but is not limited to, mouse, rat, rabbit, dog, simian, etc. The non-human mammal is relatively close to human in terms of genome composition, organ dissection, individual development, metabolic mode, and disease pathogenesis, and can be well applied to human disease pathogenesis and drug pharmacology research, etc.

[0037] (8) The animal model of insect protein food allergy of the present application is prepared by immunizing a non-human mammal with a protein allergen isolated from insect protein.

[0038] (9) Preferably, in a specific embodiment of the present application, the non-human mammal is a mouse. More preferably, in an embodiment of the present application, the mouse strain used is BALB / c.

[0039] (10) Regarding the process of immunization, including sensitization treatment and challenge treatment.

[0040] (11) In a specific embodiment of the present application, the sensitization treatment and the challenge treatment are performed by oral gavage.

[0041] (12) In a preferred embodiment of the present application, the sensitization treatment and the challenge treatment are performed by oral gavage, and the non-human mammal is systematically immunized with an insect protein allergen.

[0042] (13) In a preferred embodiment of the present application, the sensitization treatment and the challenge treatment are performed by oral gavage; in the sensitization treatment stage, cholera toxin CT is used as a mucosal immunoadjuvant, and the dose of cholera toxin CT is 10 μg per mouse per time; and the sensitization treatment is performed by oral gavage once a week for five consecutive weeks; and the challenge treatment is performed on the seventh day after the end of the sensitization treatment stage.

[0043] (14) After the sensitization induction and challenge of the animal with an insect protein allergen are completed, the inventors of the present application use various indicators of sensitization reaction to verify whether the model is successful or not, such as "body weight change", "allergic clinical symptom score", "content of allergen-specific IgE in serum", and "content of histamine in serum". It is found through verification that, after the sensitization induction and challenge with an insect protein, the mouse shows obvious allergic clinical symptoms, and the IgE level and the content of histamine in the serum of the mouse are significantly increased. In combination of these indicators, it can be seen that the inventors of the present application have successfully constructed a mouse model of insect protein food allergy by using an insect protein.

[0044] (15) The animal model of insect protein food allergy constructed by the present application can be used for the research of the pathogenesis of insect food allergy, such as the mechanism research of the intestinal mucosal immune system, the allergen-related intestinal flora and the marker differential metabolites, the cell level or the molecular level. The mechanism of the allergy (immune response) caused by the insect food is researched and explained, and a method for developing a method for reducing the allergenicity of insect protein is provided.

[0045] (16) It should be understood that, although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the description of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

[0046] (17) The series of detailed descriptions listed above are only specific descriptions of the feasible embodiments of the present application, and are not used to limit the protection scope of the present application, and any equivalent embodiments or changes made without departing from the spirit of the present application should be included in the protection scope of the present application.

[0047] Example 1

[0048] Extraction of black soldier fly protein, including the following contents:

[0049] (1) The black soldier fly bodies are washed with distilled water and 95% alcohol. Then the three kinds of insects are vacuum dried and crushed with an ultramicro grinder to obtain insect powder;

[0050] (2) Take 20g of insect powder, add 200mL of petroleum ether and n-hexane (v / v=1:3), stir at 4℃ for 2h, after standing, pour out the upper solution, then add 200mL of petroleum ether and n-hexane for degreasing, repeat three times, then centrifuge at 4℃, 8000rpm / min for 20min, take the lower precipitate and dry, which is the defatted insect powder;

[0051] (3) Add 5g of the defatted black soldier fly powder obtained in step (2) above to 100mL of distilled water, adjust the pH to 10, stir at 4℃ for 1h, then centrifuge at 4℃, 8000rpm / min for 20min, take the supernatant; add 100mL of distilled water to the lower precipitate and operate in the same way to obtain the supernatant. Combine the two times of supernatant, and desalt the supernatant at 4℃ for 48h. Freeze-dry the final extract and store at -20℃.

[0052] (4) Identification of the product: re-dissolve the target protein obtained by separation and purification in step (3) above into PBS solution for SDS-PAGE gel electrophoresis, and the electrophoretogram result is shown in Figure 5 .

[0053] (5) The protein content of the target protein PBS solution obtained above was determined by BCA method (Bicinchoninic Acid Assay), and the protein concentration was adjusted and placed in a -80°C refrigerator for standby.

[0054] Example 2

[0055] Extraction of silkworm pupa protein

[0056] (1) The silkworm pupa was washed with distilled water and 95% alcohol. Then it was vacuum dried and crushed with an ultramicro grinder to obtain insect powder;

[0057] (2) 20 g of insect powder was taken and 200 mL of petroleum ether and n-hexane (v / v = 1:3) was added, and stirred at 4°C for 2 h. After standing, the upper solution was poured out and then 200 mL of petroleum ether and n-hexane was added for degreasing. After repeating three times, centrifugation was performed at 4°C, 8000 rpm / min for 20 min, and the lower precipitate was taken and dried, which was the defatted insect powder;

[0058] (3) 5 g of the defatted silkworm pupa powder obtained in step (2) above was added to 100 mL of distilled water, and the pH was adjusted to 10. After stirring at 4°C for 1 h, centrifugation was performed at 4°C, 8000 rpm / min for 20 min, and the supernatant was taken. 100 mL of distilled water was added to the lower precipitate, and the same operation was performed to obtain the supernatant. The two times of supernatant were combined, and the supernatant was desalted at 4°C for 48 h. The final extract was freeze-dried and stored at -20°C.

[0059] (4) Identification of the product: the target protein obtained by separation and purification in step (3) above was resuspended in PBS solution for SDS-PAGE gel electrophoresis, and the electrophoretogram results are shown in Figure 5 .

[0060] (5) The protein content of the target protein PBS solution obtained above was determined by BCA method (Bicinchoninic Acid Assay), and the protein concentration was adjusted and placed in a -80°C refrigerator for standby.

[0061] Example 3

[0062] Construction of an animal model of black soldier fly protein food allergy, including the following contents:

[0063] (1) Experimental animals: the experimental animals used in the present application are BALB / c mice, purchased from VITROLIFE (Beijing) Biotechnology Co., Ltd., and raised in the animal house of Jiangnan University (free feeding, feeding environment temperature 22-25°C, humidity 40-60%) and related test operation.

[0064] (2) After one week of adaptive feeding, BALB / c mice were randomly divided into two groups according to body weight. They were given free access to water and food and were weighed weekly.

[0065] (3) Animal model construction experiment: The construction experiment process of this example is divided into "allergen sensitization treatment stage" and "allergen provocation treatment".

[0066] (4) Allergen sensitization treatment stage: Mice were gavaged twice a week for four consecutive weeks, and the gavage dose during this stage was 200 μL / mouse. Experimental group: The three experimental groups were gavaged with 200 μL of a buffer solution (PBS buffer) containing black soldier fly protein (50 mg / kg body weight) and CT (10 μg / mouse); wherein the black soldier fly protein was obtained by separation and purification in Example 1 above; CT (10 μg / mouse) is a conventional mucosal immune adjuvant cholera toxin. Blank control group: The gavage solution was 200 μL of a PBS buffer solution containing CT (10 μg / mouse).

[0067] (5) Allergen challenge: Seven days after the allergen sensitization phase, gavage challenge was performed, with a gavage challenge dose of 200 μL / animal. Experimental group: The gavage administration solution was 200 μL of PBS buffer containing black soldier fly insect protein (250 mg / kg body weight) (the gavage challenge solution did not contain CT, and the insect protein dose was 5 times the dose of the sensitization treatment); blank control group: The gavage administration solution was 200 μL of PBS buffer.

[0068] (6) Detection of allergic reaction indicators: In the examples of the present application, the allergic reaction indicators detected include "weight changes during sensitization", "allergic clinical symptom score", "serum IgE content" and "serum histamine content".

[0069] (7) Body weight changes during sensitization: During the above-mentioned allergen sensitization treatment phase, the body weight of mice was measured before each allergen gavage sensitization. The statistical results of body weight changes during sensitization are as follows: Figure 1 The mice in each group were active and their weight increased steadily, with no significant abnormal reactions. Based on the changes in the mice's bodies, the food allergy model construction method used in this patent had no significant effect on the mice's diet during the experiment, and the mice had no significant abnormal reactions before stimulation.

[0070] (8) Allergy clinical symptom scoring: 40 minutes after the above-mentioned allergen challenge treatment (i.e., 40 minutes after gavage challenge), the mice in each group were scored for allergy clinical symptoms. The scoring criteria are shown in Table 1 below. The statistical results of the allergy clinical symptom scoring are shown in Table 1. Figure 2 .from Figure 2As can be seen, compared to the control group, mice in the black soldier fly protein group developed grade 1-2 allergic symptoms, including increased hair growth, nose scratching, and decreased activity. In the mealworm protein group, half of the mice showed grade 1 allergic symptoms, while the other half did not. Therefore, based on the "allergy clinical symptom score" indicator, the mouse model constructed using black soldier fly insect protein in this application is capable of simulating a mouse model of black soldier fly protein food allergy.

[0071] Table 1

[0072]

[0073] (9) Detection of IgE content in serum: 1 hour after the above-mentioned allergen stimulation treatment "oral stimulation", blood was collected from mice in each group (blood was collected by removing the eyeballs) to measure the IgE content level in the mouse serum. This application uses an ELISA kit purchased from Nanjing Senbega Technology Co., Ltd. to detect the IgE content in the serum of each group of mice. The specific operation process is shown in the kit instructions, which will not be repeated in this invention. The specific IgE content detection results are shown in Figure 3 .from Figure 3 As can be seen in the results, the serum IgE level in the black soldier fly protein group was significantly higher than that in the blank control group, increasing by 1.34 times. Therefore, judging from the index of "serum IgE content", the allergic mouse model constructed by the black soldier fly protein in this application can simulate the mouse model of black soldier fly protein food allergy.

[0074] (10) Detection of histamine content in serum: 1 hour after the above-mentioned allergen stimulation treatment "gavage stimulation", blood was collected from mice in each group (blood was collected by removing the eyeballs) to measure the histamine content in the mouse serum. This application uses an ELISA kit purchased from Nanjing Senbega 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 instructions, which will not be repeated here. The histamine content detection results are shown in Figure 4 .from Figure 4 As can be seen in the data, the histamine content in the serum of the black soldier fly protein group (7.39±1.64ng / mL) was significantly higher than that of the blank control group (4.22±1.14ng / mL). Therefore, judging from the "serum histamine content" indicator, the allergic mouse model constructed using black soldier fly protein in this application can simulate the mouse model of black soldier fly protein food allergy.

[0075] (11) Based on the results of (7)-(10) above, the results of various allergic reaction indicators in the mouse model constructed by sensitization and stimulation treatment with black soldier fly protein in this application were significantly higher than those in the blank control group. The allergic mouse model constructed by this application using black soldier fly protein can simulate the mouse model of black soldier fly protein food allergy.

[0076] Example 4

[0077] The construction of an animal model of silkworm pupa protein food allergy includes the following:

[0078] (1) Experimental animals: The experimental animals used in this application were BALB / c mice, purchased from Weitonglihua (Beijing) Biotechnology Co., Ltd., and raised in the animal room of Jiangnan University (free diet, breeding environment temperature 22-25°C, humidity 40-60%) and related experimental operations.

[0079] (2) After one week of adaptive feeding, BALB / c mice were randomly divided into four groups according to body weight. They were given free access to water and food and were weighed weekly.

[0080] (3) Animal model construction experiment: The construction experiment process of this example is divided into "allergen sensitization treatment stage" and "allergen provocation treatment".

[0081] (4) Allergen sensitization treatment stage: Mice were gavaged twice a week for four consecutive weeks, and the gavage dose during this stage was 200 μL / mouse. Experimental group: The three experimental groups were gavaged with 200 μL of buffer solution (PBS buffer) containing silkworm pupa protein (50 mg / kg body weight) and CT (10 μg / mouse); wherein, silkworm pupa protein was obtained by separation and purification as described in Example 1; CT (10 μg / mouse) is a conventional mucosal immune adjuvant cholera toxin. Blank control group: The gavage administration solution was 200 μL of PBS buffer solution containing CT (10 μg / mouse).

[0082] (5) Allergen challenge treatment: Seven days after the allergen sensitization treatment, gavage challenge was performed, with a gavage challenge dose of 200 μL / mouse. Experimental group: The gavage administration solution was 200 μL of PBS buffer containing the allergen silkworm pupa protein (250 mg / kg body weight) (the gavage challenge solution did not contain CT, and the insect protein dose was 5 times the dose of the sensitization treatment); blank control group: The gavage administration solution was 200 μL of PBS buffer.

[0083] (6) Detection of allergic reaction indicators: In the examples of the present application, the allergic reaction indicators detected include "weight changes during sensitization", "allergic clinical symptom score", "serum IgE content" and "serum histamine content".

[0084] (7) Body weight changes during sensitization: During the above-mentioned allergen sensitization treatment phase, the body weight of mice was measured before each allergen gavage sensitization. The statistical results of body weight changes during sensitization are as follows: Figure 1The two groups of mice were active and their body weight increased steadily, and no obvious abnormal reaction was observed. According to the results of the changes in the mice, the food allergy model construction method used in the present application had no obvious effect on the diet of the mice during the experiment, and the mice had no significant abnormal reaction before the challenge.

[0085] (8) Allergic clinical symptom score: 40 min after the above-mentioned allergen challenge treatment (i.e. 40 min after the gavage challenge), the allergic clinical symptom score of each group of mice was scored, and the scoring standard is shown in Table 1 below. The statistical results of the allergic clinical symptom score are shown in Figure 2 From Figure 2 , compared with the control group, the mice in the silkworm pupa protein group showed 1-2 grade allergic symptoms, including increased hair, scratching nose and reduced activity. Therefore, from the "allergic clinical symptom score" index, the mouse model constructed by the silkworm pupa protein used in the present application can simulate the mouse model of silkworm pupa protein food allergy.

[0086] Table 2

[0087]

[0088] (9) Detection of IgE content in serum: 1 h after the above-mentioned allergen challenge treatment "gavage challenge", the blood of each group of mice was taken (enucleation of the eyeball to take blood), and the content level of IgE in the serum of each group of mice was determined. The ELISA kit purchased from Nanjing Sunbiaga Technology Co., Ltd. was used to detect the IgE content in the serum of each group of mice, and the specific operation process is described in the kit instruction manual, which will not be described herein. The results of the allergen-specific IgE content detection of each group are shown in Figure 3 From Figure 3 , it can be seen that the silkworm pupa protein group was significantly higher than the blank control group, and increased by 1.36 times. Therefore, from the "serum IgE content" index, the mouse model constructed by the silkworm pupa protein used in the present application can simulate the mouse model of silkworm pupa protein food allergy.

[0089] (10) Detection of histamine content in serum: 1 h after the above-mentioned allergen challenge treatment "gavage challenge", the blood of each group of mice was taken (enucleation of the eyeball to take blood), and the content level of histamine in the serum of each group of mice was determined. The ELISA kit purchased from Nanjing Sunbiaga Technology Co., Ltd. was used to detect the histamine content in the serum of each group of mice, and the specific operation process is described in the kit instruction manual, which will not be described herein. The results of the histamine content detection of each group are shown in Figure 4 From Figure 4As can be seen from the above, the content of histamine in the serum of the chrysalis protein group mice (6.16±0.56 ng / mL) was significantly higher than that of the blank control group (4.22±1.14 ng / mL). Therefore, from the index of "serum histamine content", the mouse model constructed by the present application using chrysalis protein can simulate a mouse model of chrysalis protein food allergy.

[0090] (11) According to the results of (7)-(10) above, in the mouse model constructed by the present application using chrysalis protein for sensitization and challenge, the results of each sensitization reaction index of chrysalis protein were significantly higher than those of the blank control group. The mouse model constructed by the present application using chrysalis protein can simulate a mouse model of chrysalis protein food allergy.

[0091] The above provided examples are not intended to limit the scope covered by the present application, and the described steps are not intended to limit the execution order thereof. The improvements of the present application made by those skilled in the art in combination with the existing common knowledge are also within the protection scope defined by the claims of the present application.

Claims

1. A method for constructing an animal model of insect protein food allergy, characterized in that: Includes the following: S1. Extracting insect protein from insects, adding the insect protein and cholera toxin to a buffer solution to obtain a sensitizing solution, and adding the insect protein to the buffer solution to obtain a provocative solution, wherein the insect protein content in the provocative solution is 4 to 6 times the insect protein content in the sensitizing solution; S2. The mice were sensitized by gavage with the sensitizing solution and the sensitization treatment was continued for 3 to 5 weeks; S3. On the 6th to 8th day after sensitization, mice were gavaged with the challenge solution; S4. After the stimulation treatment, the mice were scored for clinical allergic symptoms, and the allergenicity of the insect protein was determined based on the clinical allergic symptom scores of the mice.

2. The construction method according to claim 1, characterized in that The buffer solution is PBS buffer.

3. The construction method according to claim 1, characterized in that The mice used were BALB / c strain.

4. The construction method according to claim 1, characterized in that During sensitization treatment, the dosage of insect protein is 30 to 80 mg / kg body weight each time.

5. The construction method according to claim 1 or 4, characterized in that: During sensitization treatment, the dosage of cholera toxin was 8-12 μg per mouse each time.

6. The construction method according to claim 1, characterized in that During the stimulation treatment, the dosage of insect protein is 150-400 mg / kg body weight each time.

7. The construction method according to claim 1, characterized in that The extraction steps of insect protein are: S01. The dried insect bodies are washed with distilled water and 90% to 100% alcohol, and then dried and crushed to obtain insect powder; S02, defatting the insect powder with petroleum ether and n-hexane to prepare defatted insect protein powder; S03. Immerse the defatted insect protein powder in distilled water with a pH of 9 to 11 to extract the insect protein.

8. The construction method according to claim 1, wherein: Sensitization treatment included gavage of sensitization solution to mice 1 to 3 times per week.

9. An animal model constructed by the construction method according to any one of claims 1 to 8.

10. Use of the animal model according to claim 9 in screening drugs against insect protein allergies.

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