CN111351921A - Food sensitization animal model, construction method and application thereof - Google Patents
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Abstract
The invention provides a food allergen sensitized animal model, a construction method and application thereof. The invention carries out mouse immune reconstruction through healthy human peripheral blood cells, and carries out human immune reconstruction on an immune-deficient mouse by adopting an optimized food allergen extraction method, food allergenic protein quantity and optimized human immune cell quantity, thereby obtaining a food allergenic protein sensitized human immune reconstructed mouse model which has high success rate and can be successfully carried out by healthy human blood, and providing a simple and quick drug effect evaluation method for drug effect evaluation of allergic disease drugs.
Description
Technical Field
The invention belongs to the technical field of food safety detection technology, food safety and drug effect detection and evaluation, and particularly relates to a food sensitization animal model, a construction method and application thereof.
Background
Hypersensitivity is one of allergic diseases, and relates to food safety and the occurrence and development of various diseases, such as food allergy or food allergy of digestive system, allergic gastroenteritis and the like, inhalation sensitization can cause allergic asthma, contact sensitization can cause urticaria, eczema and the like, the pathogenesis of hypersensitivity is that allergen activates lymphocytes through TSLP, then lymphocytes release IL-4,5,13, so as to activate B cells to secrete IgE, and then immunoglobulin IgE mediates the mast cells to generate hypersensitivity immune reaction, so as to cause the allergic reaction of digestive system, respiratory system, skin or whole body, and the mechanism of the allergic reaction is shown in figure 1 (Israel E, New England Journal of Medicine, 2017, 377(10):965 976.).
Food allergen sensitization actually means that after some people eat and inject some food/allergen, the food/allergen causes a strong reaction of some tissue, some organ or even the whole body of the body, so that various dysfunctions or tissue injuries occur. It is an abnormal immune reaction produced by immune system to a specific food allergen, the immune system can produce a specific immunoglobulin to the food, when the specific immunoglobulin is combined with the food allergen, a plurality of chemical substances can be released, so that the allergic symptoms are caused, and even the serious person can cause anaphylactic shock. The mechanism of hypersensitivity reaction and inhalation sensitization, contact sensitization and blood/body fluid pathway sensitization have similar mechanism and process. The food allergen sensitization model can be used as a substitute and simulation of other multiple sensitization models.
Common food allergens are crustacean proteins, eggs and their products, milk and its products, peanuts and their products, beans and nuts, etc.
Crustacean protein allergen: as seafood products are favored by more and more people, reports of sensitization to such food allergens are increasing. Among them, shrimp allergens are attracting much attention, and 0.6% to 2.8% of patients with allergic diseases are reported to be sensitized to shrimps. At least 13 IgE binding proteins were detected in shrimp meat, but tropomyosin was identified as the only major allergen with a relative molecular mass between 34000-39000.
The allergens of eggs and products thereof: the egg allergen is one of the most common reasons for food sensitization reaction of children, and the positive rate of the egg allergen reaches 35% in food sensitization of children, and the sensitization of adults also reaches 12%. The main allergens of the proteins were ovomucoid Galdl (relative molecular mass 28000), ovalbumin Gald2 (relative molecular mass 44000), ovotransferrin Gald3 (relative molecular mass 77000) and lysozyme Gald4 (relative molecular mass 14000); the main allergen of egg yolk is Ot yolk protein (relative molecular mass 70000). In egg sensitization, egg white is reported to be more likely to cause sensitization than egg yolk, with ovomucoid as the primary allergen.
Milk and its product allergens: the milk sensitization rate of children under 2 years old is 1.6-2.8%, and 50-90% of children are tolerated before 6 years old. The main allergens of the milk are casein (Bosd 8, relative molecular mass 20000-30000), bovine serum albumin (Bosd 6, relative molecular mass 67000) and bovine immunoglobulin, wherein the immunogenicity and antigenicity of the casein are strongest. Milk antigens are relatively stable and retain their immunogenicity after conventional processing.
Peanut and its product allergen: due to the severity and high prevalence of peanut protein sensitization, this allergen has attracted considerable attention from medical institutions. Peanut sensitization is reported to account for 10% to 47% of food-induced sensitization. The main peanut allergens are Arah1 (relative molecular mass of 63500), Arah2 (relative molecular mass of 17000), other related antigens Arah3 (relative molecular mass of 60000) and Arah4 (relative molecular mass of 14000). Minor antigens are Arah6, Arah7 and actin. ELISA inhibition tests prove that Arah2 is a main antigen for causing cross reaction of peanuts with hazelnuts, almonds and the like. Different food treatment regimens have different effects on the peanut antigen, and baking has been reported to increase the content of Amhl and make IgE antibody binding epitopes more accessible for antibody binding. Although peanuts and leguminous plants have cross-reactive proteins, clinical cross-reactions are rare. Peanut sensitization is often seen in individuals sensitized to eggs, milk, walnuts and the like, but no protein which has cross-reactivity with walnuts is found. In many cases, peanut sensitization is present throughout life and can be exacerbated.
Food allergen sensitization is the induction of an immune response mediated by food allergens through IL-4,5,13 by immunoglobulin IgE, the major immunoglobulin mediating type I allergy, which presents a high affinity IgE Fc receptor (FceR I) on basophils, mast cells and dendritic cells, and a low affinity IgE Fc receptor (FceR II, i.e., CD23) on eosinophils, monocytes, macrophages, lymphocytes and platelets.
When a patient first contacts (ingests/inhales/injects, etc.) an allergen, the body activates T lymphocytes by RSLP, stimulates IL-4,5,13 secretion, and then specific IgE is synthesized by B lymphocytes. IgE can bind to FceR I on the surface of mast cells and basophils, and also bind to FceRII on the surface of eosinophils, monocytes, macrophages, lymphocytes and the like. When the body is exposed to the allergen again, it forms an antigen with IgE bound to FceR I. Only 15-20% of the FceR I needs to be crosslinked to cause a large amount of inflammatory mediators released by mast cells/basophils, thereby causing smooth muscle contraction, increased mucus secretion, increased vascular permeability and inflammatory cell infiltration. (Xia A N, World Clinical Drugs, 2010, 1: 005.).
For the treatment of IgE-mediated allergy, commonly used drugs are those that antagonize the pathway of development of hypersensitivity reactions, such as anti-TSLP antibodies, anti-IL-4 antibodies, anti-IL-5 antibodies, antibody IgE antibodies, and the like. Antibody IgE antibodies significantly reduce free IgE levels by binding to free IgE, blocking IgE binding to mast cells, eosinophils, and preventing the release of inflammatory mediators. Antibodies or inhibitors against TSLP, IL-4, IL-5, etc. may prevent the release of inflammatory mediators by reducing IgE production or inhibiting eosinophils.
The hypersensitive response antagonist can bind to free IgE in blood and interstitial fluid or reduce the production of IgE, thereby blocking or reducing the binding of IgE to the Fc epsilon RI and Fc epsilon RI; when the concentration of free IgE approaches 0, the Fc epsilon RI expression on the surfaces of basophils and mast cells can be reduced, so that the cells cannot respond to the stimulation of the allergen. In anti-IgE therapy, complexes formed by IgE and anti-IgE antibodies accumulate rapidly to more than 10 times the basal value of IgE, thus having a scavenger effect on newly incoming allergenic molecules. And can bind to and potentially down-regulate IgE-expressing B lymphocytes, and the allergen is no longer transmitted to the IgE-preferred CD23 pathway (Segal M, World Allergy organization journal, 2008, 1(10): 174-183.).
There are many animal models for evaluating the efficacy of anti-IgE pathway (TSLP, IL-4, IL-5, IgE, histamine, etc.) antibodies, and the current methods for evaluating the efficacy of anti-IgE pathway mainly include:
(1) the macaque animal sensitization model comprises the following steps: sensitization was required 31 weeks in advance (allergen extract 40ug/kg +20mg/ml alum). And (4) screening animals with successful sensitization and then administering the animal. The simulation is most similar to human body simulation, but the test period is long, the number of animals is large, and the cost is high. (Shields R L, International Archives of Allergy & Immunology, 1995, 107(1-3): 308-.
(2) Surrogate antibody surrogate model: through sensitizing a mouse, then using a Rat anti-mouse antibody as a proxy antibody of a human anti-antibody to research the drug effect, the cost of an animal model is low, but the proxy antibody (needing to be purchased or made by oneself) is used, so the model is not persuasive to be used for developing and using Biosimilar, in addition, even in the development of new drugs, only a substitute antibody of an anti-mouse target can be evaluated to carry out the summary idea verification, and the meaning is limited because the evaluated target drug cannot be directly verified. (Tumas D B, J Allergy Clin Immunol, 2001, 107(6): 1025-.
(3) Immunization of sensitized patients to reconstruct a mouse model: the immune cells of the sensitized patient are reconstituted by a heavily immunodeficient mouse, then the allergen is sensitized, and the administration of an anti-IgE pathway antibody/antagonist is carried out after sensitization. This model is more representative of the in vivo efficacy of anti-IgE pathway antibodies/antagonists, but in the case of less sensitized patients, the patient is ethically flawed by taking a large amount of blood, and if more mice are required for immune reconstitution, a smaller number of patients are not able to provide sufficient blood cell mass, and is more difficult to implement. (Pagovich O E, Journal of Allergy & Clinical Immunology, 2016, 138(6): 1652.).
(4) Hematopoietic stem cell immune reconstitution: hematopoietic stem cells are used for immune reconstruction of heavy immunodeficiency mice, and after 4 months of reconstruction, allergen sensitization is required for 8 weeks. Screening by large-dose allergen challenge is then performed, and finally, anti-IgE pathway antibody/antagonist is performed for efficacy evaluation. The method adopts hematopoietic stem cells for transplantation immune reconstruction, solves the problem of blood donors, but needs massive reconstruction, and carries out later-stage allergen stimulation, the stimulation success rate is not high, and mice with unsuccessful stimulation need to be screened and removed. The whole model has long construction period and high cost. (Burton O T, Journal of Allergy & Clinical Immunology, 2016, 139(1): 314.).
Disclosure of Invention
Because various models in the prior art have defects, the invention provides a food allergen sensitized animal model, a construction method and application thereof.
The invention aims to carry out mouse immune reconstruction through healthy human peripheral blood cells, and carry out human immune reconstruction on an immune-deficient mouse by adopting an optimized food allergen extraction method, food allergenic protein quantity and optimized human immune cell quantity to obtain a food allergenic protein sensitized human immune reconstructed mouse model which has high success rate and can be successfully carried out by healthy human blood, thereby providing a simple and rapid drug effect evaluation method for drug effect evaluation of allergic diseases (such as anti-TSLP antibody/antagonist, anti-IL-4 antibody/antagonist, IL-5 antibody/antagonist, anti-IgE antibody/antagonist, anti-histamine antagonist and the like).
The invention enables the immunodeficient mouse to have a human immune system by reconstructing the immunodeficient mouse. In the invention, anticoagulated whole blood of healthy volunteers is extracted, healthy human lymphocytes are separated and obtained, the healthy human lymphocytes are injected into severe immunodeficiency mice, and the human immune system of the severe immunodeficiency mice is reconstructed.
The mice after the reconstruction of the human immune system are sensitized and stimulated. Injecting the separated food allergen protein into a human immune reconstituted mouse body to carry out sensitization construction on the human immune reconstituted mouse; and injecting food allergen protein into the human immune reconstructed mice to excite the sensitization reaction.
The mice are subjected to blood collection before modeling, sensitization and excitation to detect IgE, histamine, CD45 and CD23 and evaluate sensitization symptoms such as weight, pruritus, hair, action state and respiration condition of the mice.
Obtaining a food-sensitized animal test mouse after screening, applying an anti-IgE antibody or other allergic disease medicines to the food-sensitized animal test mouse, collecting blood of the mouse after application, detecting the change of allergic symptom indexes such as IgE, histamine, CD45, CD23 and the like, and evaluating the drug effect of the allergic medicine by using the food-sensitized animal model.
A food-sensitized animal model characterized in that human immune system reconstitution of immunodeficient mice is performed using normal healthy human PBMCs.
The food allergen-sensitized animal model described above, wherein the food allergen-sensitized animal model constructing step includes: 1) human immune system reconstitution of immunodeficient mice using normal healthy human PBMCs; 2) food sensitizing protein is used for sensitizing and exciting sensitization reaction of human immune reconstituted mice.
The food-sensitized animal model as described above, characterized in that the food-sensitized animal model constructing step comprises: 1) injecting normal healthy human PBMC and food sensitizing protein into an immunodeficiency mouse, and feeding; 2) mice that have completed human immune reconstitution are challenged with a sensitization response using a food sensitizing protein.
The food-sensitized animal model according to the above, wherein the food-sensitized animal model is constructed by the step 1) of injecting a mixture of PBMCs of a normal healthy human and a food-sensitized protein into an immunodeficient mouse, wherein the number of PMBC of the normal healthy human is 5 × 106A/only 3 × 107One/one; the amount of the food sensitizing protein is 100 ug/piece to 300 ug/piece.
The food-sensitized animal model is characterized in that the food-sensitized animal model construction step 2) uses food-sensitized protein to excite the sensitization reaction of the mice which have completed human immune reconstitution, and the amount of the food-sensitized protein is 100 ug/mouse to 300 ug/mouse.
A method for preparing a food sensitized animal model is characterized in that the food sensitized animal model construction step comprises 1) intraperitoneal injection of 2 × 10 to an immunodeficient mouse7Feeding mixed solution of PBMC of normal and healthy people and food-sensitized protein of 100 ug/person; 2) mice that had completed human immune reconstitution were challenged with 100 ug/mouse of food sensitizing protein for a total of 4 challenges weekly.
The preparation method of the food sensitized animal model is characterized in that the food sensitized protein is crustacean protein, eggs and egg products, milk and milk products, peanut and peanut products, beans and nuts.
The method for preparing the food-sensitized animal model is characterized in that the food-sensitized protein is peanut and peanut products.
Use of a food-sensitized animal model as described above for the evaluation of the efficacy of a drug for the treatment of allergic diseases.
The use of the food-sensitized animal model as described above is characterized in that the food-sensitized animal model is used for evaluation of drug efficacy of an anti-TSLP antibody/antagonist, an anti-IL-4 antibody/antagonist, an IL-5 antibody/antagonist, an anti-IgE antibody/antagonist, an anti-histamine antagonist and the like.
NCG mice (NOD-Prkdcem 26Il2rgem26/Nju, NOD/ShiLtJNju mice knockout Prkdc and IL2RG genes), Nanjing university institute of model animals, SPF grade, female, 6-8 weeks old; ZJ507, Shanghai Mitai Juno Biotechnology Co., Ltd; Xolair/Omalizumab (507 OR), NOVARTIS); intravenous injection of human immunoglobulin, specification: 1 ml/piece, 50mg/ml, Shandong Taibang biological products, Inc.; lymphocyte separation medium, specification: 250 ml/bottle, cat No.: 85-2001, Shanghai Huajing Biotech, Inc.; mouse anti-human CD45 FITC, specification: 1 ml/count, Shanghai Mitaijuno Biotech limited; PE Mouse Anti-Human CD3, Specification: 100 TESS, cat number: 555340, BDPharmingen; human histamine assay kit, cat No.: E01H0013, shanghai blue-based biotechnology limited; IgE detection Kit Immunoglobulin E (IgE) Human ELISA Kit, cat # ab178659, abcam.
Drawings
FIG. 1: schematic diagram of allergy mechanism;
FIG. 2: a standard curve chart for peanut protein content determination;
FIG. 3: human immunization reconstructs the symptom map of the mouse.
Detailed Description
The invention is further illustrated by taking a peanut sensitized animal model as an example.
The following examples are merely illustrative of the present invention and should not be construed as limiting thereof.
Example 1: preparation of food-sensitized protein
The preparation method of the peanut allergenic protein comprises the following steps: placing raw semen Arachidis Hypogaeae into oven, baking at 100 deg.C for 5min, and cooling at room temperature. Grinding the roasted peanut kernel to obtain peanut powder, weighing 25g of peanut powder, dissolving in 20mmol/L Tris buffer (pH7.2), stirring at 23 deg.C for 2h, high speed centrifuging for 30min, coarse filtering the supernatant in a negative pressure device, and sterile filtering with a disposable 0.45um needle filter.
Peanut protein concentration was determined using Bradford method: preparing a group of bovine serum albumin solutions with the concentrations of 0.10mg/ml, 0.08mg/ml, 0.06mg/ml, 0.04mg/ml, 0.02mg/ml and 0mg/ml respectively, and measuring the absorbance of the solutions to obtain a standard curve of the protein concentration to the absorbance. And obtaining the concentration of the unknown protein sample according to the standard curve.
Determination of peanut protein content: a group of standard substances and peanut protein are added into a 96-hole enzyme label plate, wherein each standard substance and peanut protein are 50 ul/hole, and then 200 ul/hole of the prepared Coomassie brilliant blue solution is added. Standing for 10min, measuring absorbance (wavelength 595 nm) of standard and sample with enzyme labeling instrument, inputting relevant parameters with OD value as ordinate and standard protein content as abscissa in SoftMax Pro6.6.2 software, selecting Linear, and automatically generating standard curve chart (shown in FIG. 2).
The equation is Y = A + BX, R2>0.95, and the greater the value of R2 of the absorbance-concentration relationship curve, the better the linearity of the measured absorbance and concentration. Inputting a formula in the software, and automatically substituting the numerical value into the formula so as to calculate the protein concentration (mg/ml) of the peanut protein.
Example 2: PBMC preparation
Pbmc (peripheral blood mononuclear cells) comprising lymphocytes, monocytes, dendritic cells and other small numbers of cells.
Approximately 400ml of anticoagulated whole blood was collected from healthy volunteers and subjected to lymphocyte separation using room temperature lymphocyte separation medium.
The method comprises the steps of subpackaging lymphocyte separation liquid into a plurality of 15ml centrifuge tubes at 7 ml/tube, centrifuging at 1000rpm for × min, centrifuging and throwing liquid attached to the tube wall to the bottom, placing the centrifuge tubes in a 37 ℃ water bath for keeping out of the sun for later use, placing PBS (phosphate buffer solution) in a 37 ℃ water bath for later use, diluting human peripheral blood with pre-warmed PBS according to the ratio of 1:2, uniformly mixing, inclining the centrifuge tubes at an angle of 45 degrees, placing the diluted blood above the interface of the lymphocyte separation liquid at a position close to the interface of the lymphocyte separation liquid, slowly adding the diluted blood into the upper layer of the interface of the lymphocyte separation liquid at 5 ml/tube, balancing the centrifuge tubes with a balance, placing the centrifuge tubes into a centrifuge, setting the centrifuge program to 2000rpm × min at room temperature, setting the centrifugal speed to the lowest value, dividing the centrifuge tubes into 4 layers after centrifugation is finished, setting the upper layer to be plasma, blood diluent and most platelets at the upper layer, setting the lower layer to be red cells and granulocytes, setting the middle layer to be cell layering liquid, setting the supernatant layer to be 6330 min, setting the centrifugal layer to be gray layer as a gray layer, setting the centrifugal layer to be a straw to be a gray layer, slightly inserting gray single nucleus cell, slightly inserting a gray layer, setting the PBS along the tube wall, adding the PBS, setting the centrifugal layer, setting the centrifugal tube to be another PBS, setting the centrifugal tube to be a lifting program, setting the centrifugal value, setting.
Example 3: establishment of food sensitization animal model
1. Sensitization in food-sensitized animal models:
the lymphocytes isolated in example 2 were resuspended in PBS (phosphate buffered saline) and the lymphocyte density was adjusted to 1 × 107Each 0.5ml of the total extract is 2 × 107Each 0.5ml of the total extract is 3 × 107Each 0.5ml, 5 × 106Each 0.5ml, preferably 2 × 1070.5ml (lymphocyte density screening results are shown in Table 1); mixing the above lymphocytes with 100ug/0.5ml, 200 ug/0.5ml, 300 ug/0.5ml extractive solution of isolated peanut protein, preferably 100ug/0.5ml (see Table 2 for peanut protein concentration screening results); the mixed solution containing the healthy human lymphocytes and the isolated peanut protein was injected 2-sidedly into each of severe immunodeficiency mice (NCG mice, 250ul per one-sidedly).
Measuring histamine content in mouse serum using a human metabolism-related enzyme-linked immunosorbent assay (ELISA) using a commercially available human histamine ELISA kit; CD45 and CD3 are detected by adopting a direct labeling method and a flow cytometry method, mouse peripheral blood is labeled by adopting a direct labeling flow antibody and two-color fluorescence, and the expression of various genes of PBMC in CD45 or CD3 is directly detected by . Detecting and counting the single-cell liquid flow form by using a BD flow cytometer, wherein a cell marker tracer substance used for detection is a fluorescent marker.
TABLE 1 results of lymphocyte Density screening test
Third week after PBMC transplantation, mice in the high dose group died.
2. Stimulation of food-sensitized animal models:
after injecting mixed solution containing healthy human lymphocytes and peanut protein isolate into severe immunodeficiency mice, injecting 100ug/0.5ml, 200 ug/0.5ml and 300 ug/0.5ml peanut protein isolate extracting solution into abdominal cavity every week, injecting peanut protein isolate extracting solution into two sides of abdominal cavity, exciting food sensitized animal model, and continuously injecting for 3 weeks.
Each peanut sensitization response was fasted 8 hours prior to stimulation to maximize the uptake of peanut antigen by the mice.
Orbital blood sampling (whole blood sampling after dosing) and weighing and sensitization symptom evaluation were performed on all mice before modeling (i.e., before PBMC transplantation and peanut extract injection), after modeling (i.e., before drug administration (4 weeks after transplantation and peanut sensitization)), after drug administration (i.e., 30 minutes after drug administration), respectively.
Meanwhile, after 4 weeks of sensitization, approximately 20ul of anticoagulated whole blood was collected from all mice in the eye orbit, and Human CD3 and Human CD45 were detected by FACS Canto ii.
The double positive ratio of Human CD3 and Human CD45 in peripheral blood of the sensitized mice is detected, and whether the transplantation of the Human PBMC is successful in the mice is confirmed.
And (3) determining whether the establishment of the peanut sensitized mouse disease model is successful by comparing the content changes of total IgE and histamine in mouse serum before and after modeling.
TABLE 2 peanut protein concentration screening test results
In the high dose group, mice died after four challenges.
Combining the test results, 100 ug/peanut protein dose is selected to stimulate the sensitization reaction of the human immune reconstituted mouse.
Example 4 use of food-sensitized animal models
Detection of drug efficacy of anti-IgE antibody:
the efficacy of anti-IgE antibodies was evaluated using the food-sensitized animal model constructed in the above example. The therapeutic effect of ZJ507 (anti-IgE antibody) and Omalizumab (commercially available anti-IgE antibody) on diseases caused by IgE, whether the therapeutic effect can bind to free IgE in sensitized mice and relieve and treat sensitization symptoms of mice can be confirmed by comparing the content change of free IgE and histamine in the serum of mice before and after administration. Measuring histamine content in mouse serum using a human metabolism-related enzyme-linked immunosorbent assay (ELISA) using a commercially available human histamine ELISA kit; the Immunoglobulin E (IgE) Human ELISA Kit provided by abcam is selected to detect the total IgE in the serum of the experimental Human immune reconstituted mouse.
TABLE 3 results of histamine and IgE content detection before and after administration of anti-IgE antibody
The results show that the anti-IgE antibodies ZJ507 and the Omalizumab can obviously reduce the content of histamine and IgE in a sensitized animal model and can obviously relieve inflammatory symptoms caused by sensitization. The test animal models provided in examples 1, 2 and 3 can be used for evaluating the efficacy of allergic diseases.
Claims (11)
1. A food-sensitized animal model characterized in that human immune system reconstitution of immunodeficient mice is performed using normal healthy human PBMCs.
2. The food sensitized animal model according to claim 1, characterized in that said food sensitized animal model building step comprises: 1) human immune system reconstitution of immunodeficient mice using normal healthy human PBMCs; 2) food sensitizing protein is used for sensitizing and exciting sensitization reaction of human immune reconstituted mice.
3. The food sensitized animal model according to claim 2, characterized in that said food sensitized animal model building step comprises: 1) injecting normal healthy human PBMC and food sensitizing protein into an immunodeficiency mouse, and feeding; 2) mice that have completed human immune reconstitution are challenged with a sensitization response using a food sensitizing protein.
4. The food-sensitized animal model according to claim 3, characterized in that the food-sensitized animal model construction step 1) comprises injecting a mixture of normal healthy human PBMCs and food-sensitized proteins into an immunodeficient mouse, wherein the number of normal healthy human PMBC is 5 × 106A/only 3 × 107One/one; the amount of the food sensitizing protein is 100 ug/piece to 300 ug/piece。
5. The food-sensitized animal model according to claim 3, characterized in that the food-sensitized animal model construction step 2) performs sensitization reaction excitation on mice that have completed human immune reconstitution with food-sensitized proteins in an amount of 100 ug/mouse to 300 ug/mouse.
6. A method for preparing a food sensitized animal model is characterized in that the food sensitized animal model construction step comprises 1) intraperitoneal injection of 2 × 10 to an immunodeficient mouse7Feeding mixed solution of PBMC of normal and healthy people and food-sensitized protein of 100 ug/person; 2) mice that had completed human immune reconstitution were challenged with 100 ug/mouse of food sensitizing protein for a total of 4 challenges weekly.
7. The method of preparing a food-sensitized animal model according to claim 6, characterized in that said food-sensitized protein is crustacean protein, eggs and egg products, milk and milk products, peanut and peanut products, beans, nuts.
8. The method of preparing a food-sensitized animal model according to claim 7, wherein said food-sensitized protein is peanut and peanut products.
9. Use of a food sensitized animal model according to claim 1 or claim 6 for the evaluation of the efficacy of a medicament for the treatment of allergic diseases.
10. Use of a food sensitized animal model according to claim 9, characterized in that said food sensitized animal model is used for the evaluation of the efficacy of anti-TSLP antibody/antagonist, anti-IL-4 antibody/antagonist, IL-5 antibody/antagonist, anti-IgE antibody/antagonist, anti-histamine antagonist.
11. Use of a food sensitized animal model according to claim 10 for the evaluation of the efficacy of anti-IgE antibodies/antagonists.
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