Construction method of animal model for infant nutrition rice flour food allergy

Abstract

The invention relates to the field of prevention and treatment of physical allergy of infants, in particular to a construction method of an animal model for allergy of infant nutrition rice flour food. The method includes: 1, adaptively feeding experimental animals in multiple groups randomly according to weights; 2, in the first day to the seventh day, different test adjuvants are used for experiments on each group of experimental animals, one group of experimental animals is subjected to negative blank control without adjuvants, and the other groups are divided into different small groups according to different test substances; the animals are fed regularly and quantitatively every day, a negative blank group is fed with a basic liquid feed agent without adjuvant matching, and the other groups are fed with liquid feed with the same amount containing 0.5 mL of immunologic adjuvant solution; 3, allergy excitation is conducted at 9: 00 a.m. on the eighth day, a blank group is fed with 5 g of purified water, the other groups are fed with 5 g of infant nutrition rice flour liquid feed, animal clinical allergic symptoms are observed, and blood and tissue samples are collected; killing the animal, and collecting a blood sample; the experimental animal is preferably an SPF (specific pathogen free) level female Wistar rat with the weight of 45-65 g and the age of 3 weeks.

Description

Technical Field

[0001] The invention relates to the technical field of prevention and treatment of infant allergy to real objects, and in particular to a method for constructing an animal model of infant allergy to nutritious rice flour. Background Art

[0002] Food allergy is a major public health problem that has attracted global attention due to its high prevalence. Approximately 5% of adults and 8% of children worldwide suffer from food allergy, and the prevalence has been steadily increasing over the past three decades.

[0003] According to epidemiological surveys, the incidence of food allergies among Chinese children increased from 3.5% in 1999 to 11.1% in 2019. In addition, 8% of children in the United States and 6.7% of the total population in Canada are affected by food allergies. The associated economic, emotional, and safety burdens brought about by the above diseases are enormous.

[0004] According to the pathogenesis, there are four main types of allergic reactions (I-IV). Type I is a reaction to allergenic foods mediated by immunoglobulin (Ig) E, types II and III involve IgG-mediated reactions, and type IV is attributed to the reaction of allergen-specific T cells. Among them, common food allergies belong to type I allergic reactions. In the past few decades, about 8% of children and 5% of adults worldwide have allergic reactions to food, and the incidence of food allergies is on the rise. Most foods can cause allergies, but the eight major types of allergic foods jointly promulgated by the Food and Agriculture Organization of the United Nations and the World Health Organization mainly include milk, peanuts, nuts, wheat, eggs, sesame, fish and crustacean aquatic products.

[0005] The occurrence of food allergies is related to many factors. In terms of the age of onset, patients with egg and milk allergies are mostly infants and young children, and the allergic symptoms gradually disappear with age. The difference in the prevalence of food allergies is also closely related to the local dietary conditions. Studies have shown that among schoolchildren in southern my country, doctors diagnosed the prevalence of crustacean allergies in urban areas as significantly higher than in rural areas. The geographical environment is another factor that affects the rate of food allergies. The allergic foods of allergic people in Western countries are mainly peanuts and nuts, while in Eastern countries they are mostly fish and crustacean products. In addition, the incidence of food allergies caused by genetics has also attracted much attention. Studies have found that parents with a history of peanut allergies have a 7-fold increased risk of their children suffering from peanut allergies.

[0006] Food allergy is an adverse stress allergic reaction to food in allergic patients or sensitive people, which is a serious threat to the health of the majority of food allergy patients. Food allergy is clinically manifested as adverse symptoms of skin (allergic dermatitis and urticaria, etc.), oral and digestive tract (slurred speech, oral swelling, stomach pain, abdominal pain and diarrhea, etc.), respiratory tract (sneezing, runny nose, asthma, shortness of breath and difficulty breathing, etc.), cardiovascular system (palpitations and accelerated heartbeat, etc.) and nervous system (mental confusion and unconsciousness, etc.). Severe food allergies can cause shock or even death in allergic patients.

[0007] Most food allergens are highly resistant to heat, pH and gastrointestinal enzymes. They are defined as specific ingredients in food that can be recognized by allergen-specific immune cells, triggering specific immune responses and leading to allergic symptoms. To date, the number of allergens named by the World Health Organization (WHO) / International Union of Immunological Societies (IUIS) Allergen Nomenclature Group is as high as 1,062, of which 399 are food-derived allergens. They are further divided into about 60% plant-derived allergens and 40% animal-derived allergens.

[0008] Infant and young children's nutritional rice cereal is a supplementary food suitable for infants and young children over 6 months old, which is made of rice as the main raw material, with appropriate amounts of nutritional enhancers and (or) other auxiliary materials added. Rice accounts for about 95% of the raw materials and occupies a major position in the raw and auxiliary materials of infant and young children's nutritional rice cereal. Infant and young children's rice cereal is a food specially designed for infants and young children. The nutrients usually include protein, carbohydrates, fat, vitamins and minerals, which are essential for the growth and development of infants. Because this rice cereal is easy to digest, it can help infants better absorb these nutrients and promote their healthy growth. More importantly, infant and young children's rice cereal does not contain the eight major allergens, which means that it will hardly cause allergic reactions. This is undoubtedly a very good choice for those infants and young children who are prone to allergies. Therefore, compared with other foods, the probability of allergies in infant and young children's rice cereal is relatively low, and it is a good food source to reduce or relieve allergic symptoms. However, in recent years, food allergies caused by rice have occurred frequently, and the incidence of allergies to infant nutritional rice cereal is still increasing, which has had many adverse effects on the development and health of infants and young children. Therefore, evaluating the allergenicity of infant nutritional rice cereal and reducing its allergenicity is of extremely important practical significance for preventing infant consumers from suffering from allergic diseases and promoting their healthy growth.

[0009] The rising prevalence of food allergies and the problems brought about by various new protein resources to food allergy risk management have made food allergenicity evaluation the basis for rationally controlling food allergy risks and ensuring food safety. Currently, there are many methods that can be used to evaluate food allergenicity, but different methods have different advantages and disadvantages and scope of application.

[0010] The application of bioinformatics in allergenicity evaluation is mainly concentrated in the field of genetically engineered proteins. It cannot directly determine whether the target protein is an allergen, but predicts its allergenicity by analyzing the homology and cross-reactivity between the target protein and known allergens; antibody-based immunological analysis methods rely on the preparation of antibodies and the separation and purification of antigens, and complex food processing techniques can lead to changes in the structure of allergen proteins, causing them to lose their original antigenic activity, which can easily lead to false positive or false negative test results; nucleic acid-based PCR analysis technology detects DNA rather than protein, and the test results cannot truly reflect the allergenicity of the food, and there are many factors that affect the amplification efficiency. , the difference in amplification efficiency between actual samples and standard samples can affect the cycle threshold, making it difficult to meet high-precision quantification; mass spectrometry detection technology has the characteristics of high sensitivity, strong specificity, good accuracy and high throughput, but the expensive mass spectrometry equipment and the need for professionals make the detection cost and labor cost of this method high, which also limits its wide application in food allergen detection; evaluating the resistance of target proteins to gastrointestinal digestion through in vitro simulated digestion experiments is also one of the common methods for evaluating allergenicity, but in vitro simulated digestion shows positive, that is, it has anti-digestion properties, and it cannot directly determine that the target protein is allergenic, and further verification is required through serological experiments or biological models. Although overall, in vitro allergenicity evaluation methods have the advantages of low cost and high efficiency, in vitro evaluation methods can only partially reflect the allergenicity of the target protein, especially they cannot accurately evaluate the allergenicity of potential primary allergens and cannot simulate the complex environment in the human body well. Therefore, in vivo evaluation methods are still necessary to control the allergenic risks of new foods in the future. Therefore, more reliable food allergenicity evaluation can be obtained in human experiments, and commonly used evaluation methods are skin irritation tests and dietary challenge experiments. However, their development and application are also greatly restricted due to their high difficulty, high risk, high cost, and ethical issues. Currently, only a few hospitals have the conditions and qualifications to conduct human experiments.

[0011] Compared with in vivo evaluation in humans, animal model evaluation has the advantages of abundant animal sources, simple methods and no harm to the human body. It is also valuable in understanding the complex immunological and pathophysiological mechanisms involved in the occurrence and development of food allergic reactions. Therefore, animal models are often used to evaluate the potential allergenicity of allergens.

[0012] Animal model evaluation methods, commonly used animal models include rats, dogs and pigs. When choosing, it is necessary to clarify the operating characteristics, limitations and reliability in different environments. At present, rats are the most studied, because people have a better understanding of the rat immune system, and they are also tools for many immune-related studies. In addition, rats are the most commonly used experimental animals in food safety evaluation studies. The results of other food safety studies can serve as the basis for allergenicity studies of related substances. There have been reports on the use of BN rats to evaluate food allergies, but there have been no reports on the use of Wistar rats to establish animal models of food allergens. In addition, there are three commonly used sensitization routes in animal experiments for allergenicity evaluation: oral gavage, intraperitoneal injection and free feeding. Intraperitoneal injection can study the intrinsic allergenicity of proteins, but this route is not the natural route for humans to ingest food allergens. Therefore, oral gavage and free feeding are often used. The advantage is that their exposure routes are most relevant to human contact with food allergens.

[0013] In addition, existing food allergenicity testing technologies and allergic reaction potential assessment methods cannot truly reflect allergenicity or risk, and some methods are too expensive and are not suitable for low-allergenicity product testing. Therefore, an assessment method that can truly reflect the sensitization of allergens has broad application value in the fields of scientific research and production, and is of great significance to promoting related basic research and technological applications of allergies.

[0014] At present, in the research on animal allergenicity models, the animal test cycle of most researchers is often 4-6 weeks or more after the weaning period, while infant nutritional rice cereal is a supplementary food for infants and young children, and its feeding cycle is 6 to 36 months. Therefore, the existing animal modeling has the disadvantages of a long cycle and cannot fully simulate the food allergy reactions in infancy. In addition, the digestive system of infants and young children is not yet mature, and the absorption and processing of food ingredients are different from those of adult animals, which will also affect the distribution of potential allergens in the body and the manifestation of immune responses. In addition, the immune system of infants and young children is not yet fully developed. Compared with adult animals or humans, they may react differently to allergens and show different allergic reaction mechanisms from adults. Traditional animal models are mostly used for adult animals and may not accurately reflect the allergic reactions of infants and young children. In addition, the allergic mechanism of infant groups is very complex, and the allergic reactions of infants and young children are different in different living environments and age stages, because some allergens may not cause reactions in adult animal models, but in real life, they may cause severe allergies after ingestion by infants and young children. For example, rice protein may not be recognized as an allergen in some adult mouse animal models, but it can cause allergic symptoms in infants and young children. Therefore, animal experiments that match the environment and age are needed to verify the data to ensure that the data is accurate. According to relevant reports, 2 to 3 weeks for rats is approximately equivalent to 1 year for humans. Therefore, it is not possible to use a conventional animal test cycle. The animal test cycle must be controlled within the required range and the animal model must have the characteristics of accurately responding to infant allergic symptoms, so as to simulate the changes in food allergic reactions in infancy and young children, thereby establishing the optimal animal model. In addition, BN rats have a high similarity with humans in immune system and physiological characteristics, especially in the mechanism and immune pathways of allergic reactions, but they are expensive and have high animal experiment costs. Wistar rats have low economic costs and can meet the requirements of food allergy sensitization experiments. Therefore, establishing a humanized animal model with simulated allergic characteristics in infancy can accurately study the specific mechanism of allergic reactions caused by nutritional rice cereal in infants and young children, which is conducive to in-depth research on the pathogenesis and diagnosis and treatment of the corresponding diseases without bringing treatment risks to clinical subjects. The animal cost is low and the effect is excellent. It is of great significance to provide a theoretical basis for the clinical accurate diagnosis of nutritional rice cereal allergy in infants and young children. Summary of the invention

[0015] The purpose of the present invention application is to address the deficiencies of the prior art and solve the above-mentioned technical problems. The first purpose is to provide a method for constructing an animal model of infant nutritional rice flour food allergy and the application of the animal model.

[0016] The second purpose of the present invention is to use the animal model constructed by the construction method in the study of allergic diseases caused by allergens, wherein the purpose of the study is non-disease diagnosis or treatment;

[0017] The third purpose of the present invention is to provide a method for screening potential substances that are anti-infant rice flour allergy, the method comprising: applying relevant candidate substances to an animal model constructed by the above-mentioned construction method, and detecting the allergic reaction indicators of the animal model.

[0018] The fourth purpose of the present invention is to provide, on the one hand, the use of the animal model constructed by the above-mentioned construction method in screening drugs for preventing food allergies to infant nutritional rice flour or in experimental research on food allergies to infant nutritional rice flour and infant complementary food.

[0019] To achieve the above object, the present invention adopts the following technical scheme: a method for constructing an animal model of infant nutritional rice flour food allergy, comprising the following steps:

[0020] Step 1: Experiments were conducted after the experimental animals were adaptively fed. The animals were randomly divided into several groups according to their body weight and were allowed to drink water and eat freely during the period.

[0021] Step 2: On the 1st to 7th day, each group of experimental animals was tested with different adjuvants, one group of experimental animals received a negative blank control without adjuvant, and the remaining groups were divided into different groups according to different test substances; during the test, each animal was fed 5g of food at 9:00 am and 4:00 pm every day, the negative blank group was fed with a basic liquid feed without adjuvant, 2.4g + 2.6g water, with a dilution consistency of 48%, and the remaining groups were fed with the same amount of liquid feed containing 0.5mL of immune adjuvant solution;

[0022] Step 3: Allergy stimulation was performed at 9:00 am on the 8th day. The blank group was fed with 5g of pure water, and the other groups were fed with 5g of infant nutrition rice flour liquid feed for allergy stimulation. The animals were fasted for 12h before feeding, and the clinical allergic symptoms of the animals were observed within 30min-1h, and the corresponding blood and tissue samples were collected;

[0023] Then, the animals were killed and blood samples were collected;

[0024] The experimental animals are preferably 3-week-old female Wistar rats of SPF grade weighing 45 g to 65 g.

[0025] Furthermore, the animals were randomly divided into 11 groups according to their body weight, with 6 experimental animals in each group. One group was tested with lipopolysaccharide, which was administered orally; the other groups were fed freely, and the test substances were normal saline, lipopolysaccharide, β-glucan, chitosan, wolfberry polysaccharide, astragalus polysaccharide, manno-oligosaccharide, mannose, ginseng stem and leaf saponin, and zinc hydroxide.

[0026] Furthermore, the formula of the basic liquid feed is: casein, L-cystine, cellulose, xanthan gum, maltodextrin, sucrose, soybean oil, mixed minerals, mixed vitamins, and choline bitartrate.

[0027] Furthermore, the dosage of the above-mentioned test substance and the basic liquid feed configuration are as follows:

[0028] 1. Preparation of immune adjuvant

[0029] All immune adjuvant solutions were prepared before use, with specific concentrations as follows: lipopolysaccharide concentration was 0.10 mg / mL; β-glucan, chitosan, wolfberry polysaccharide, and astragalus polysaccharide concentration was 0.244 g / mL; manno-oligosaccharide, mannose, and riboflavin concentration was 10.0 mg / mL; ginseng stem and leaf saponin concentration was 3.0 mg / mL; and zinc hydroxide concentration was 2.0 mg / mL.

[0030] 2. Basic liquid feed configuration

[0031] Each mouse was fed 5g of food each time. The blank group was fed with 2.4g of basic liquid feed + 2.6g of water. The preparation method was as follows: accurately weigh 2.4g of liquid feed, mix it evenly with 2.6g of water, and then prepare and stir it to make a basic liquid feed with a consistency of 48%. The other groups were fed the same amount of liquid feed containing 0.5mL of immune adjuvant solution.

[0032] Furthermore, the blood sampling and testing steps in step 3 are as follows: each rat is anesthetized by intraperitoneal injection of 10% (w / v) chloral hydrate solution at 0.3g / 100g (body weight), and then the abdominal skin is disinfected with alcohol, the abdominal skin and muscles are cut with surgical scissors, and the rat abdominal aorta blood is collected under sterile conditions in a negative pressure blood collection tube and an EDTA anticoagulant tube, respectively. After collection, the blood is centrifuged at 3500rpm / min for 15-20min at 4°C, and the supernatant of both tubes is placed in a cryopreservation tube, and stored in a -80°C refrigerator after liquid nitrogen cold extraction for serum and plasma specific antibodies and histamine determination.

[0033] The above-mentioned specific antibodies are specific IgG, specific IgE, mast cell protease mMCP-1, tryptase MCT and histamine HIS.

[0034] Furthermore, the detection steps of specific antibodies are:

[0035] The obtained serum and plasma were measured in the same manner as described in step 3 according to the instructions of the ELISA kit, as follows:

[0036] 1. Take out the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes, seal the remaining strips with self-sealing bags and store them back at 4°C, set standard wells and sample wells, add 50μL of standard of different concentrations to each standard well, and then add 50μL of the sample to be tested to the sample well; do not add to the blank well; if the sample needs to be diluted, dilute the sample with the sample diluent provided with the kit as required;

[0037] 2. Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 30 minutes. Do not add biotin-labeled antibody to the standard wells or blank wells.

[0038] 3. Discard the liquid and pat dry on absorbent paper, then fill each well with washing solution (350 μL), let it stand for 1 min, then discard the washing solution and pat dry on absorbent paper. Repeat this process 5 times.

[0039] 4. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard well and sample well, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 30 minutes;

[0040] 5. Discard the liquid and pat dry with absorbent paper. Fill each well with washing solution (350 μL). Let stand for 1 min, then discard the washing solution and pat dry with absorbent paper. Repeat this process 5 times.

[0041] 6. Add 50 μL of substrate A (chromogen A: H2O2) and B (chromogen B: TMB) to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well. Use an ELISA reader to measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0042] Furthermore, the large-dose nutritious rice flour liquid feed for infants and young children is a nutritious rice flour liquid feed for infants and young children prepared by mixing and stirring 5 g of nutritious rice flour for infants and young children with 10 g of water.

[0043] Beneficial effects of the present invention: After extensive and in-depth research, infants and young children will have enhanced tolerance to food allergies as they grow and age, so the animal model constructed by this method is adopted. The animal model obtained by the present invention overcomes the inability of the in vitro evaluation method to fully simulate the complex immune response of the body to infant nutritional rice flour, and compared with the existing food allergy animal model, it overcomes the disadvantage that the animal modeling cycle is long and cannot fully simulate the food allergy reaction in the infant period. It can be used to evaluate the allergenicity of infant nutritional rice flour and various infant complementary foods, fundamentally reducing the risk of infant rice flour-like food allergies in infant groups, and can simulate the food allergy characteristics of infant groups, with strong clinical relevance, and can be used in experimental research on infant rice flour and infant complementary food allergies, and provide a research model for clinical research on infant food allergies or drug screening against infant food allergies, providing an important choice and basis for the clinicalization of basic research. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Attached Figure 1 This is a grouped clinical symptom scoring diagram for Example 3 of the present invention.

[0045] Attached Figure 2 This is a diagram of body temperature in groups according to Example 3 of the present invention.

[0046] Attached Figure 3 This is a diagram of the diarrhea situation in the group of Example 3 of the present invention.

[0047] Attached Figure 4 This is a graph showing the IgG content in groups according to Example 3 of the present invention.

[0048] Attached Figure 5 This is a diagram of the IgE content in groups according to Example 3 of the present invention.

[0049] Attached Figure 6 This is a graph showing the mMCP-1 content in groups according to Example 3 of the present invention.

[0050] Attached Figure 7 This is a graph of the trypsin (MCT) content in group 3 of Example 3 of the present invention.

[0051] Attached Figure 8 This is a diagram of HIS content in groups according to Example 3 of the present invention. DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0053] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercial products. The infant nutrition rice flour used in the present invention is a certain brand of commercially available 1st-stage infant nutrition rice flour, and the test substances are all purchased from reagent suppliers.

[0054] Embodiment 1:

[0055] This example provides an animal model construction and sensitization stimulation method, the specific steps are as follows:

[0056] Construction of sensitized animal model:

[0057] (1) Animal requirements:

[0058] Wistar rats, SPF grade, 3 weeks old, female, 45 g to 65 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0059] (2) Test substances

[0060] Normal saline, lipopolysaccharide, β-glucan, chitosan, wolfberry polysaccharide, astragalus polysaccharide, manno-oligosaccharide, mannose, ginseng stem and leaf saponin, zinc hydroxide.

[0061] (3) Animal grouping

[0062] The experimental animals were randomly divided into 11 groups (6 animals in each group) according to their body weight, namely: negative blank group, lipopolysaccharide group, β-glucan group, chitosan group, wolfberry polysaccharide group, astragalus polysaccharide group, manno-oligosaccharide group, mannose group, riboflavin, ginseng stem and leaf saponin group, and zinc hydroxide group.

[0063] (4) Formula of basic liquid feed

[0064] The formula of the basic liquid feed is: casein, L-cystine, cellulose, xanthan gum, maltodextrin, sucrose, soybean oil, mixed minerals, mixed vitamins, choline bitartrate;

[0065] (5) Dosage of test substance and liquid feed requirements:

[0066] 1) Preparation of immune adjuvant

[0067] All immune adjuvant solutions were prepared before use, with specific concentrations as follows: lipopolysaccharide concentration was 0.10 mg / mL; β-glucan, chitosan, wolfberry polysaccharide, and astragalus polysaccharide were 0.244 g / mL; manno-oligosaccharides, mannose, and riboflavin were 10.0 mg / mL; ginseng stem and leaf saponins were 3.0 mg / mL; and zinc hydroxide was 2.0 mg / mL.

[0068] 2) Liquid feed preparation

[0069] Each mouse was fed 5 g of food each time. The blank group was fed 2.4 g of basic liquid feed + 2.6 g of water (accurately weigh 2.4 g of basic liquid feed, mix it with 2.6 g of water, then prepare and stir it to make a basic liquid feed with a consistency of 48%), and the other groups were fed the same amount of liquid feed containing 0.5 mL of immune adjuvant solution.

[0070] (6) Sensitization

[0071] The large-dose infant nutritional rice flour liquid feed sensitization challenge is an infant nutritional rice flour liquid feed prepared by mixing and stirring 5g of infant nutritional rice flour in 10g of water.

[0072] (7) Establishment of sensitization model

[0073] Purchased Wistar rats were housed in metabolic cages, one rat per cage, with an indoor temperature of 26±2°C, a humidity of 50% to 60%, and a day-night cycle of 12:12h, for 7 days of adaptive feeding.

[0074] On the 1st to 7th day, each group of experimental animals was experimented with different test adjuvants, one group of experimental animals received a negative control without adjuvant, and the remaining groups were divided into different groups according to different test substances; during the experiment, each animal was fed 5g of food at 9:00 am and 4:00 pm every day, the negative blank group was fed with a basic liquid feed without adjuvant (2.4g + 2.6g water, 48% consistency), and the remaining groups were fed with the same amount of liquid feed containing 0.5mL of immune adjuvant solution.

[0075] Allergy stimulation was performed at 9:00 am on the 8th day. The blank group was fed 5g of pure water, and the other groups were fed 5g of infant nutritional rice flour liquid feed for allergy stimulation (fasting for 12h before feeding). The clinical allergic symptoms of the animals were observed within 30min-1h, and corresponding blood and tissue samples were collected.

[0076] Embodiment 2:

[0077] In the examples of the present application, the detected allergic reaction indicators include allergic clinical symptom scores, body temperature, diarrhea, and specific antibodies IgG, IgE, mast cell protease (mMCP-1), tryptase (MCT) in serum and histamine (HIS) in plasma.

[0078] (1) Allergy clinical symptom score

[0079] According to Table 1, the clinical allergic symptoms of rats were evaluated within 30 minutes to 1 hour after allergic stimulation of rats, and the allergic reaction symptoms were recorded and observed. The observation was continued and real-time monitoring and scoring were performed.

[0080] Table 1 Clinical symptom scores

[0081]

[0082]

[0083] (2)Body temperature

[0084] Within 30 minutes to 1 hour after allergic stimulation, the rectal temperature of the rats was measured multiple times using a rectal thermometer to observe changes in the rats' body temperature.

[0085] (3) Diarrhea

[0086] Within 30min-1h after allergic stimulation, the occurrence of diarrhea in rats was observed, and the incidence rate of diarrhea in each group of mice was calculated.

[0087] (4) Determination of specific antibodies (IgG, IgE) in serum

[0088] The obtained serum was assayed according to the ELISA kit instructions, as follows:

[0089] 1) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes, seal the remaining strips with a ziplock bag and return them to 4°C, set up standard wells and sample wells, and add 50μL of different concentrations of standard wells to each well. Then add 50μL of the sample to be tested to the sample well; do not add to the blank well. If the sample needs to be diluted, dilute the sample with the sample diluent provided with the kit as required.

[0090] 2) Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with a sealing film, and incubate at 37°C in a water bath or incubator for 30 min. Do not add biotin-labeled antibody to standard wells or blank wells.

[0091] 3) After discarding the liquid, pat dry with absorbent paper and fill each well with washing solution (350μL). After standing for 1 minute, shake off the washing solution and pat dry with absorbent paper. Repeat this washing process 5 times (you can also use a plate washer to wash the plate).

[0092] 4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 30 min.

[0093] 5) Discard the liquid and pat dry with absorbent paper, fill each well with washing solution (350 μL), let it stand for 1 min, then shake off the washing solution and pat dry with absorbent paper. Repeat this process 5 times (you can also use a plate washer to wash the plate).

[0094] 6) Add 50 μL of substrate A (chromogen A: H2O2) and B (chromogen B: TMB) to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well. Use an ELISA reader to measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0095] (5) Mast cell protease (mMCP-1)

[0096] The obtained serum was assayed according to the ELISA kit instructions, as follows:

[0097] 1) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes, seal the remaining strips with a ziplock bag and return them to 4°C, set up standard wells and sample wells, and add 50μL of different concentrations of standard wells to each well. Then add 50μL of the sample to be tested to the sample well; do not add to the blank well. If the sample needs to be diluted, dilute the sample with the sample diluent provided with the kit as required.

[0098] 2) Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with a sealing film, and incubate at 37°C in a water bath or incubator for 30 min. Do not add biotin-labeled antibody to standard wells or blank wells.

[0099] 3) After discarding the liquid, pat dry with absorbent paper and fill each well with washing solution (350μL). After standing for 1 minute, shake off the washing solution and pat dry with absorbent paper. Repeat this washing process 5 times (you can also use a plate washer to wash the plate).

[0100] 4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 30 min.

[0101] 5) Discard the liquid and pat dry with absorbent paper, fill each well with washing solution (350 μL), let it stand for 1 min, then shake off the washing solution and pat dry with absorbent paper. Repeat this process 5 times (you can also use a plate washer to wash the plate).

[0102] 6) Add 50 μL of substrate A (chromogen A: H2O2) and B (chromogen B: TMB) to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well. Use an ELISA reader to measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0103] (6) Tryptase (MCT)

[0104] The obtained serum was assayed according to the ELISA kit instructions, as follows:

[0105] 1) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes, seal the remaining strips with a ziplock bag and return them to 4°C, set up standard wells and sample wells, and add 50μL of different concentrations of standard wells to each well. Then add 50μL of the sample to be tested to the sample well; do not add to the blank well. If the sample needs to be diluted, dilute the sample with the sample diluent provided with the kit as required.

[0106] 2) Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with a sealing film, and incubate at 37°C in a water bath or incubator for 30 min. Do not add biotin-labeled antibody to standard wells or blank wells.

[0107] 3) After discarding the liquid, pat dry with absorbent paper and fill each well with washing solution (350μL). After standing for 1 minute, shake off the washing solution and pat dry with absorbent paper. Repeat this washing process 5 times (you can also use a plate washer to wash the plate).

[0108] 4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 30 min.

[0109] 5) Discard the liquid and pat dry with absorbent paper, fill each well with washing solution (350 μL), let it stand for 1 min, then shake off the washing solution and pat dry with absorbent paper. Repeat this process 5 times (you can also use a plate washer to wash the plate).

[0110] 6) Add 50 μL of substrate A (chromogen A: H2O2) and B (chromogen B: TMB) to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well. Use an ELISA reader to measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0111] (7) Histamine (HIS)

[0112] The obtained plasma was measured according to the ELISA kit instructions, as follows:

[0113] 1) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes, seal the remaining strips with a ziplock bag and return them to 4°C, set up standard wells and sample wells, and add 50μL of different concentrations of standard wells to each well. Then add 50μL of the sample to be tested to the sample well; do not add to the blank well. If the sample needs to be diluted, dilute the sample with the sample diluent provided with the kit as required.

[0114] 2) Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with a sealing film, and incubate at 37°C in a water bath or incubator for 30 min. Do not add biotin-labeled antibody to standard wells or blank wells.

[0115] 3) After discarding the liquid, pat dry with absorbent paper and fill each well with washing solution (350μL). After standing for 1 minute, shake off the washing solution and pat dry with absorbent paper. Repeat this washing process 5 times (you can also use a plate washer to wash the plate).

[0116] 4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 30 min.

[0117] 5) Discard the liquid and pat dry with absorbent paper, fill each well with washing solution (350 μL), let it stand for 1 min, then shake off the washing solution and pat dry with absorbent paper. Repeat this process 5 times (you can also use a plate washer to wash the plate).

[0118] 6) Add 50 μL of substrate A (chromogen A: H2O2) and B (chromogen B: TMB) to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well. Use an ELISA reader to measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0119] (8) Data processing

[0120] The experimental results were expressed as mean ± standard deviation. One-way analysis of variance was performed using SPSS, and a significant difference was considered when P < 0.05.

[0121] Embodiment 3:

[0122] According to the animal model construction method in Example 1 and the experimental method in Example 2, the specific experimental results and analysis are as follows:

[0123] 1. Clinical symptom score

[0124] When the body is stimulated by a certain allergen, a series of clinical allergic reactions will occur. The test scored the clinical symptoms of Wistar rats after allergy stimulation. The results are as follows: Figure 1 shown.

[0125] After the rats were stimulated, Figure 1 It was found that except for the blank group, rats in other groups showed clinical symptoms to varying degrees. Figure 1 As shown, the blank group showed no signs of allergic symptoms, while some rats in the lipopolysaccharide group developed more severe allergic symptoms, with one rat experiencing respiratory distress allergy. In addition, rats in the other groups scratched their noses, rubbed their heads, and even experienced swelling in their eyes and mouths, as well as increased respiratory rates. Compared with the blank group and the other groups, the allergy symptom scores of the rats in the lipopolysaccharide group were significantly increased.

[0126] The results showed that when lipopolysaccharide was used as an immune adjuvant to induce allergy in rats, the rats had the highest allergic symptom score, which triggered obvious allergic reactions. The rat sensitization model constructed by the method described in the article was successful.

[0127] 2. Rat body temperature index

[0128] The experiment statistically analyzed the body temperature of Wistar rats after allergic stimulation. The results are as follows: Figure 2 shown.

[0129] Studies have shown that when rats show clinical allergic symptoms, their body temperature drops rapidly and their activity is greatly reduced.

[0130] Depend on Figure 2 It can be seen that the body temperature of the blank group was normal, with an average body temperature of 38.08°C; except for the blank group, the body temperatures of the other groups were all decreased, namely 36.98°C, 37.40°C, 37.23°C, 37.37°C, 37.43°C, 37.50°C, 37.25°C, 37.47°C, 37.70°C and 37.70°C. Compared with the blank group and the other groups, the body temperature of the mice in the lipopolysaccharide group showed the most serious downward trend, and there was a very significant difference with the blank group rats (P < 0.01).

[0131] The results showed that when lipopolysaccharide was used as an immune adjuvant to induce allergy in rats, the body temperature of the rats dropped most significantly and the average body temperature was the lowest, which triggered an obvious allergic reaction. The rat sensitization model constructed by the method described in the article was successful.

[0132] 3. Diarrhea in rats

[0133] The experiment was conducted to statistically analyze the diarrhea of ​​Wistar rats after allergic stimulation. The results are as follows: Figure 3 shown.

[0134] like Figure 3 As shown, the blank group had no diarrhea symptoms, while the other groups had diarrhea to varying degrees; compared with the blank group, the diarrhea rates of the other groups were 66.6%, 33.3%, 33.3%, 33.3%, 33.3%, 50.0%, 33.3%, 33.3%, 50.0% and 33.3%, respectively. Compared with the blank group and the other groups, the diarrhea of ​​the rats in the lipopolysaccharide group was the most serious, and there was a significant difference between the rats in the blank group (P < 0.01). The results showed that when lipopolysaccharide was used as an immune adjuvant to induce rat allergy, the rats had the highest diarrhea rate, which triggered an obvious allergic reaction. The rat sensitization model constructed by the method described in the article was successful.

[0135] 4. Analysis of rat serum specific antibodies

[0136] The test analyzed the specific antibodies IgG and IgE in rat serum. The results are as follows: Figure 4-5 shown.

[0137] The content of IgG and IgE in the body is one of the important indicators for measuring food allergies. Allergies are mainly type I allergic reactions mediated by IgE, and IgE-mediated allergic reactions will inevitably cause increased expression of IgE and IgG.

[0138] The test analyzed the specific antibodies IgG and IgE in the serum of Wistar rats. The results are as follows Figure 4-5 As shown. The results showed that the serum IgG and IgE levels in the blank group were very low and almost unchanged, indicating that the blank group mice did not have an allergic reaction. After feeding mice with different adjuvants, the serum IgG and IgE levels of rats increased to varying degrees. Compared with the blank group and the other groups, the serum IgG and IgE levels of mice in the lipopolysaccharide group were significantly increased, and there was a very significant difference with the blank group rats (P < 0.01), indicating that the immune system in the rats played a role, causing the body to be in a sensitized state under the induction of allergens, and producing IgE and IgG antibodies that mediate allergic reactions, resulting in an increase in serum IgG and IgE levels. The results showed that when lipopolysaccharide was used as an immune adjuvant to induce rat allergy, the serum IgG and IgE levels were the highest, which triggered an obvious allergic reaction. The rat sensitization model constructed by the method described in the article was successful.

[0139] 5. Analysis of rat serum mast cell protease (mMCP-1)

[0140] The experiment analyzed rat serum mMCP-1, and the results were as follows Figure 6 shown.

[0141] mMCP-1 is an important inflammatory mediator released by basophils or mast cells after degranulation occurs after activation. It can be used as an important indicator to reflect the strength of mast cell degranulation. Figure 6 It can be seen that the serum mMCP-1 content of the blank group was very low and almost unchanged, indicating that the blank group mice did not have an allergic reaction. After feeding mice with different adjuvants, the serum mMCP-1 content of rats increased to varying degrees. Compared with the blank group and the other groups, the serum mMCP-1 content of mice in the lipopolysaccharide group was significantly increased, and there was a very significant difference with the blank group rats (P < 0.01). The results showed that lipopolysaccharide was used as an immune adjuvant to induce rat allergy, and the serum IgG and IgE levels were the highest, which triggered obvious allergic reactions. The rat sensitization model constructed by the method described in the article was successful.

[0142] 6. Analysis of rat serum tryptase (MCT)

[0143] The test analyzed serum tryptase (MCT) of Wistar rats, and the results are shown in Table 7.

[0144] When the body is stimulated by a certain allergen, a series of clinical allergic reactions will occur. Figure 7 It can be seen that after feeding mice with different adjuvants, the serum MCT content of rats increased to varying degrees. Compared with the blank group and the other groups, the serum mMCP-1 content of mice in the lipopolysaccharide group increased significantly, and there was a very significant difference with the blank group rats (P < 0.01). The results showed that lipopolysaccharide was used as an immune adjuvant to induce rat allergy, and the serum IgG and IgE content was the highest, which triggered obvious allergic reactions. The rat sensitization model constructed by the method described in the article was successful.

[0145] 7. Analysis of histamine (HIS) in rat plasma

[0146] The experiment analyzed HIS in the plasma of Wistar rats. The results are as follows Figure 8 shown.

[0147] Histamine is widely present in body tissues. When body tissues are damaged or allergic reactions occur, histamine is released, which leads to increased capillary permeability and even local edema.

[0148] Depend on Figure 8 It can be seen that after feeding mice with different adjuvants, the HIS content in rat plasma increased to varying degrees. Compared with the blank group and the other groups, the HIS content in the lipopolysaccharide group was significantly increased, and there was a very significant difference with the blank group rats (P < 0.01). The results showed that when lipopolysaccharide was used as an immune adjuvant to induce rat allergy, the plasma HIS content was the highest, which triggered an obvious allergic reaction. The rat sensitization model constructed by the method described in the article was successful.

[0149] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing an animal model of infant nutritional rice flour food allergy, characterized in that: It includes the following steps: Step 1: Experiments were conducted after the experimental animals were adaptively fed. The animals were randomly divided into several groups according to their body weight and were allowed to drink water and eat freely during the period. Step 2: On the 1st to 7th day, each group of experimental animals was tested with different adjuvants, one group of experimental animals received a negative blank control without adjuvant, and the remaining groups were divided into different groups according to different test substances; during the test, each animal was fed 5g of food at 9:00 am and 4:00 pm every day, the negative blank group was fed with a basic liquid feed without adjuvant, 2.4g + 2.6g water, with a dilution consistency of 48%, and the remaining groups were fed with the same amount of liquid feed containing 0.5mL of immune adjuvant solution; Step 3: Allergy stimulation was performed at 9:00 am on the 8th day. The blank group was fed with 5g of pure water, and the other groups were fed with 5g of infant nutrition rice flour liquid feed for allergy stimulation. The animals were fasted for 12h before feeding, and the clinical allergic symptoms of the animals were observed within 30min-1h, and the corresponding blood and tissue samples were collected; Then, the animals were killed and blood samples were collected; The experimental animals are preferably 3-week-old female Wistar rats of SPF grade weighing 45 g to 65 g.

2. The method for constructing an animal model of infant nutritional rice flour food allergy according to claim 1, characterized in that: The animals were randomly divided into 11 groups according to their body weight, with 6 animals in each group. One group was given lipopolysaccharide by oral gavage; the other groups were given free-flowing food, and the test substances were normal saline, lipopolysaccharide, β-glucan, chitosan, wolfberry polysaccharide, astragalus polysaccharide, manno-oligosaccharide, mannose, ginseng stem and leaf saponin, and zinc hydroxide.

3. The method for constructing an animal model of infant nutritional rice flour food allergy according to claim 1, characterized in that: The formula of basic liquid feed is: casein, L-cystine, cellulose, xanthan gum, maltodextrin, sucrose, soybean oil, mixed minerals, mixed vitamins, and choline bitartrate.

4. The method for constructing an animal model of infant nutritional rice flour food allergy according to claims 2 and 3, characterized in that: The dosage of the test substance and the preparation method of the basic liquid feed are as follows: 1) Preparation of immune adjuvant All immune adjuvant solutions were prepared before use, with specific concentrations as follows: lipopolysaccharide concentration was 0.10 mg / mL; β-glucan, chitosan, wolfberry polysaccharide, and astragalus polysaccharide concentration was 0.244 g / mL; manno-oligosaccharide, mannose, and riboflavin concentration was 10.0 mg / mL; ginseng stem and leaf saponin concentration was 3.0 mg / mL; and zinc hydroxide concentration was 2.0 mg / mL. 2) Basic liquid feed configuration Each mouse was fed 5g of food each time. The blank group was fed with 2.4g of basic liquid feed + 2.6g of water. The preparation method was as follows: accurately weigh 2.4g of liquid feed, mix it evenly with 2.6g of water, and then prepare and stir it to make a basic liquid feed with a consistency of 48%. The other groups were fed the same amount of liquid feed containing 0.5mL of immune adjuvant solution.

5. The method for constructing an animal model of infant nutritional rice flour food allergy according to claim 1, characterized in that: The blood sampling and testing steps in step 3 are as follows: each rat is anesthetized by intraperitoneal injection of 10% chloral hydrate solution at a body weight of 0.3g / 100g, and then the abdominal skin is disinfected with alcohol, the abdominal skin and muscles are cut with surgical scissors, and the rat abdominal aorta blood is collected under sterile conditions in a negative pressure blood collection tube and an EDTA anticoagulant tube, respectively. After collection, centrifuge at 3500rpm / min at 4°C for 15-20min, and the supernatant of both tubes is taken and placed in a cryopreservation tube, and stored in a -80°C refrigerator after liquid nitrogen cold extraction for serum and plasma specific antibodies and histamine determination.

6. The method for constructing an animal model of infant nutritional rice flour food allergy according to claim 5, characterized in that: The specific antibodies are specific IgG, specific IgE, mast cell protease mMCP-1, tryptase MCT, and histamine is HIS.

7. The method for constructing an animal model of infant nutritional rice flour food allergy according to claims 5 and 6, characterized in that: The detection steps of the specific antibody are: The obtained serum and plasma were measured in the same manner as described in step 3 according to the instructions of the ELISA kit, as follows: 1) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes, seal the remaining strips with self-sealing bags and store them back at 4°C, set standard wells and sample wells, add 50μL of standard of different concentrations to each standard well, and then add 50μL of the sample to be tested to the sample well; do not add to the blank well; if the sample needs to be diluted, dilute the sample with the sample diluent provided with the kit as required; 2) Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with a sealing film, and incubate at 37°C in a water bath or incubator for 30 minutes. Do not add biotin-labeled antibody to the standard wells or blank wells. 3) Discard the liquid and pat dry on absorbent paper, then add 350 μL of washing solution to each well, let it stand for 1 min, then discard the washing solution and pat dry on absorbent paper. Repeat this process 5 times; 4) Except for the blank wells, add 100 μL of horseradish peroxidase-labeled detection antibody to each well of the standard well and sample well, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 30 minutes; 5) Discard the liquid and pat dry with absorbent paper, add 350 μL of washing solution to each well, let it stand for 1 min, then discard the washing solution and pat dry with absorbent paper, repeat this process 5 times; 6) Add 50 μL of substrate color developer A: H2O2 and color developer B: TMB to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm using an enzyme reader within 15 min.

8. The method for constructing an animal model of infant nutritional rice flour food allergy according to claim 1, characterized in that: The preparation method of the infant nutritional rice flour liquid feed in step 3 is as follows: 5g of infant nutritional rice flour is prepared by mixing and stirring with 10g of water.

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