CN115211404B - Construction method of pumpkin seed protein sensitized mouse model - Google Patents

Abstract

The application discloses a construction method of a pumpkin seed protein sensitized mouse model, and belongs to the field of animal medicine. The construction method of the model comprises the following steps: repeatedly leaching the pumpkin seed degreasing powder with leaching buffer solution for multiple times to obtain protein liquid. And dialyzing the protein liquid, and performing vacuum freeze drying to obtain freeze-dried protein powder. Re-dissolving the freeze-dried protein powder in distilled water to obtain freeze-dried protein solution. Mixing the freeze-dried protein solution with an aluminum hydroxide adjuvant for emulsification to obtain a sensitization solution. A plurality of 3-4 week old BALB/c mice were acclimatized for 3 weeks, and each mice was intraperitoneally injected with 150. Mu.L of a sensitizing solution on days 21, 28, 35, 42, 49, 56, respectively. Each mouse was perfused with 8mg-10mg of pumpkin seed protein buffer on day 62. The model not only can comprehensively represent typical characteristics of human pumpkin seed protein allergy, but also can provide multiple blood sampling opportunities and a large amount of allergy serum, provides technical support for safety evaluation of foods/medicines, and lays a foundation for food allergy mechanism research.

Description

Construction method of pumpkin seed protein sensitized mouse model

Technical Field

The application relates to the technical field of animal medicine, in particular to a construction method of a pumpkin seed protein sensitized mouse model.

Background

Pumpkin seeds are plant seeds and nuts favored by consumers, the protein content of the pumpkin seeds is up to 35%, and the ratio of essential amino acids in the protein is similar to the amino acid pattern required by human bodies. Pumpkin seed protein is an ideal basic raw material in food processing, and has been widely used in the production of soy sauce, beverage, biscuits, ham sausage and other foods. However, the pumpkin seed protein also has sensitization risk, and researches show that the pumpkin seed protein has cross sensitization characteristics with seed storage proteins such as sesame, walnut, buckwheat seed and the like. Allergic patients can cause symptoms such as dizziness, emesis, facial and pharyngeal edema after taking pumpkin seed protein, and asthma and death when severe.

In order to ensure food safety, the generation mechanism and control strategy of the pumpkin seed protein allergy are required to be researched, and the sensitization animal model is a basic tool for deeply researching the allergy mechanism. Therefore, it is urgent to construct an animal model which can comprehensively represent typical characteristics of human pumpkin seed protein allergy and can provide a large amount of allergy serum to meet research requirements.

Disclosure of Invention

The embodiment of the application aims to construct an animal model which can comprehensively express typical characteristics of human pumpkin seed protein allergy and can provide a large amount of allergy serum to meet research requirements.

The embodiment of the application provides a construction method of a pumpkin seed protein sensitized mouse model, which comprises the following steps:

repeatedly leaching the pumpkin seed degreasing powder with leaching buffer solution for multiple times to obtain protein solution;

dialyzing the protein liquid, and performing vacuum freeze drying to obtain freeze-dried protein powder;

re-dissolving the freeze-dried protein powder in distilled water to obtain freeze-dried protein solution;

mixing and emulsifying the freeze-dried protein solution and an aluminum hydroxide adjuvant to obtain a sensitization solution;

a plurality of 3-4 week old BALB/c mice are adaptively fed for 3 weeks, and 150 mu L of the sensitization liquid is respectively injected into each of the mice from the abdominal cavity on days 21, 28, 35, 42, 49 and 56;

each mouse was perfused with 8mg-10mg of pumpkin seed protein buffer on day 62.

In one possible implementation, before the step of repeatedly leaching the pumpkin seed defatted powder with the leaching buffer solution multiple times to obtain the protein solution, the method further comprises:

taking pumpkin seed kernels and liquid nitrogen according to the ratio of 2:1, grinding in a grinder to obtain pumpkin seed emulsion;

mixing the pumpkin seed emulsion with a fat solvent for multiple times, centrifuging and precipitating to obtain a pumpkin seed degreasing precipitate;

and (3) airing the pumpkin seed degreasing precipitate in a fume hood to obtain the pumpkin seed degreasing powder.

In one possible implementation, the weight to volume ratio of the pumpkin seed defatted flour to the leaching buffer, in g/mL, is 1:8.

in one possible implementation, 50mmol/L Tris-HCl,200mmol/L NaCl,0.1mmol/L EDTA and 1mmol/L DTT are included in the leaching buffer, and the pH of the buffer is 7.4.

In one possible implementation manner, the freeze-dried protein powder is obtained by vacuum freeze-drying after the protein solution is dialyzed, and specifically comprises the following steps:

dialyzing the protein solution by using a dialysis bag with a diameter of 16mm and a molecular weight cut-off of 7 kDa;

and carrying out vacuum freeze drying on the dialyzed protein liquid to obtain the freeze-dried protein powder.

In one possible implementation manner, the freeze-dried protein powder is redissolved in distilled water to obtain a freeze-dried protein solution, which specifically includes:

the freeze-dried protein powder and distilled water are mixed according to the ratio of 1: re-dissolving in the weight-volume ratio of 0.22 to obtain freeze-dried protein solution.

In one possible implementation manner, the mixing and emulsifying the freeze-dried protein solution and the aluminum hydroxide adjuvant to obtain the sensitization solution specifically includes:

mixing the freeze-dried protein solution with an aluminum hydroxide adjuvant according to a ratio of 10:1, and mixing and emulsifying the mixture according to the volume ratio to obtain the sensitization liquid.

One or more technical solutions provided in the embodiments of the present application at least have the following technical effects or advantages:

the embodiment of the application provides a construction method of a pumpkin seed protein sensitized mouse model, which comprises the following steps: repeatedly leaching the pumpkin seed degreasing powder with leaching buffer solution for multiple times to obtain protein liquid. And dialyzing the protein liquid, and performing vacuum freeze drying to obtain freeze-dried protein powder. Re-dissolving the freeze-dried protein powder in distilled water to obtain freeze-dried protein solution. Mixing the freeze-dried protein solution with an aluminum hydroxide adjuvant for emulsification to obtain a sensitization solution. A plurality of 3-4 week old BALB/c mice were acclimatized for 3 weeks, and each mice was intraperitoneally injected with 150. Mu.L of a sensitizing solution on days 21, 28, 35, 42, 49, 56, respectively. Each mouse was perfused with 8mg-10mg of pumpkin seed protein buffer on day 62. According to the construction method of the pumpkin seed protein sensitized mouse model, the pumpkin seed protein sensitized mouse model is successfully constructed by exciting the pumpkin seed protein sensitized mouse by carrying out intraperitoneal injection of the sensitized liquid and the gastric lavage pumpkin seed protein buffer liquid for a small number of times, and the model mouse can well simulate typical characteristics of human pumpkin seed protein allergy. Meanwhile, the sensitization state of the mice is prolonged through low-dose long-term sensitization in the model construction process, so that the blood sampling times of the mice are increased, the blood sampling amount is increased, the sensitization serum amount is increased, and further the requirement of subsequent research can be met.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments of the present application will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a diagram of a solvent control experimental scheme provided in an embodiment of the present application;

fig. 2 is a schematic diagram of an experimental scheme of an adjuvant control group according to an embodiment of the present application;

FIG. 3 is a schematic diagram of a short-term sensitization group experiment according to an embodiment of the present application;

FIG. 4 is a graph showing a low dose long-term sensitization group experimental protocol provided in an embodiment of the present application;

FIG. 5 is a graph showing the weight change of mice (solvent control-solvent control, adjuvant control, short term sensitization, long term sensitization) provided in the examples of the present application;

FIG. 6 shows the spleen index change of mice according to the present application;

FIG. 7 shows the thymus index variation of mice according to the present application;

FIG. 8 is a graph showing the change in rectal temperature of a mouse according to an embodiment of the present application;

FIG. 9 is a graph showing clinical symptom scores of allergy in mice provided by an embodiment of the present application;

FIG. 10 is a measurement of total serum IgE content of mice provided in the examples of the present application (different letters represent two groups of significant differences, p < 0.05);

FIG. 11 shows the measurement of specific IgE content in mouse serum according to the examples of the present application;

FIG. 12 is a chart showing the measurement of histamine content in mouse serum according to an embodiment of the present application;

FIG. 13 is a graph showing the determination of total IgE and histamine content in six blood collection procedures in mice provided by the examples of the present application;

FIG. 14 shows the measurement of IL-4 (Th 2 type cytokine) content in mouse serum according to the present application;

FIG. 15 shows the measurement of IL-13 (Th 2 type cytokine) content in mouse serum according to the present application;

FIG. 16a is a colon slice (20-fold eyepiece) of a mouse in a solvent control group provided by an example of the present application;

FIG. 16b is a colon slice (20-fold eyepiece) of a mouse in an adjuvant control group provided by an example of the present application;

FIG. 16c is a colon slice (20-fold eyepiece) of a mouse in a short-term sensitization group provided by an example of the present application;

fig. 16d is a colon slice (20-fold eyepiece) of a mouse in a low dose long-term sensitization group provided by an example of the present application.

Detailed Description

The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are some, but not all embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

The embodiment of the application provides a construction method of a pumpkin seed protein sensitized mouse model, which comprises the following steps:

step 101: repeatedly leaching the pumpkin seed degreasing powder with leaching buffer solution for multiple times to obtain protein liquid. Specifically, the pumpkin seed defatted powder is repeatedly leached for three times by leaching buffer solution to obtain protein liquid

Step 102: dialyzing the protein liquid, and performing vacuum freeze drying to obtain freeze-dried protein powder. In practical application, dialysis can reduce damage of high-salt solution to heart and cerebral vessels and other organs of mice.

Step 103: re-dissolving the freeze-dried protein powder in distilled water to obtain freeze-dried protein solution.

Step 104: mixing the freeze-dried protein solution with an aluminum hydroxide adjuvant for emulsification to obtain a sensitization solution.

Step 105: a plurality of 3-4 week old BALB/c mice were acclimatized for 3 weeks, and each mice was intraperitoneally injected with 150. Mu.L of the sensitization solution on days 21, 28, 35, 42, 49, 56, respectively. Specifically, animal models commonly used for studying the mechanism of food sensitization are BALB/C mice, C3H/HeJ mice, wistar rats, BN rats and the like. In comparison, the BALB/c mouse has the advantages of strong susceptibility, high antibody production rate, low price and the like, so the BALB/c mouse is suitable for constructing a sensitization model. In addition, pumpkin seed protein allergy belongs to Immunoglobulin E (Immunoglobulin E) mediated I-type food allergy reaction, the content of Th2 cytokines such as IL-4, IL-13 and the like in serum of a patient suffering from the disease is high, and BALB/c mice can well simulate the reaction of immune cells of a human body, and release the Th2 cytokines such as IL-4, IL-13 and the like after sensitization, so that the sensitization of sensitizers can be accurately measured in an experiment.

Step 106: each mouse was perfused with 8mg-10mg of pumpkin seed protein buffer on day 62. In practice, each mouse was perfused with 400. Mu.L-500. Mu.L of 20mg/mL pumpkin seed protein buffer on day 62. Specifically, the preparation method of the pumpkin seed protein buffer solution used during gastric lavage excitation is that a proper amount of freeze-dried protein powder is taken and dissolved in water, and the freeze-dried protein powder is fully and uniformly shaken.

The embodiment of the application provides a construction method of a pumpkin seed protein sensitized mouse model, which comprises the following steps: repeatedly leaching the pumpkin seed degreasing powder with leaching buffer solution for multiple times to obtain protein liquid. And dialyzing the protein liquid, and performing vacuum freeze drying to obtain freeze-dried protein powder. Re-dissolving the freeze-dried protein powder in distilled water to obtain freeze-dried protein solution. Mixing the freeze-dried protein solution with an aluminum hydroxide adjuvant for emulsification to obtain a sensitization solution. A plurality of 3-4 week old BALB/c mice were acclimatized for 3 weeks, and each mice was intraperitoneally injected with 150. Mu.L of a sensitizing solution on days 21, 28, 35, 42, 49, 56, respectively. Each mouse was perfused with 8mg-10mg of pumpkin seed protein buffer on day 62. According to the construction method of the pumpkin seed protein sensitized mouse model, the pumpkin seed protein sensitized mouse model is successfully constructed by exciting the pumpkin seed protein sensitized mouse by carrying out intraperitoneal injection of the sensitized liquid and the gastric lavage pumpkin seed protein buffer liquid for a small number of times, and the model mouse can well simulate typical characteristics of human pumpkin seed protein allergy. Meanwhile, the sensitization state of the mice is prolonged through low-dose long-term sensitization in the model construction process, so that the blood sampling times of the mice are increased, the blood sampling amount is increased, the sensitization serum amount is increased, and further the requirement of subsequent research can be met.

Further, before the pumpkin seed defatted powder is repeatedly leached by the leaching buffer solution for a plurality of times to obtain the protein liquid, the method further comprises the following steps:

taking pumpkin seed kernels and liquid nitrogen according to the ratio of 2:1, grinding in a grinder to obtain pumpkin seed emulsion; mixing pumpkin seed emulsion with a fat solvent for multiple times, centrifuging and precipitating to obtain a pumpkin seed degreasing precipitate; and (3) airing the defatted pumpkin seed precipitate in a fume hood to obtain the defatted pumpkin seed powder. Specifically, the pumpkin seed degreasing powder is prepared by the following method:

taking pumpkin seed kernels and liquid nitrogen according to the ratio of 2:1, grinding in a grinder to obtain pumpkin seed emulsion;

pumpkin seed emulsion and acetone were mixed in a ratio of 1:10, magnetically stirring at 4 ℃ for degreasing for 2 hours, and centrifuging at 8000rpm for 20min to obtain primary precipitate;

the primary precipitate was taken with diethyl ether in g/mL according to 1:10, magnetically stirring at 4 ℃ for degreasing for 2 hours, and centrifuging at 8000rpm for 20 minutes to obtain secondary precipitation;

the secondary precipitate was admixed with acetone in g/mL according to a ratio of 1:10, magnetically stirring at 4 ℃ for degreasing for 2 hours, and centrifuging at 8000rpm for 20min to obtain three precipitates;

three precipitations were combined with diethyl ether in g/mL according to 1:10, magnetically stirring at 4 ℃ for degreasing for 2 hours, and centrifuging at 8000rpm for 20min to obtain four-time precipitation;

and (5) drying the four-time sediment in a fume hood to obtain the pumpkin seed degreasing powder.

Through respectively dissolving pumpkin seed chyme with acetone and diethyl ether twice for fat in the pumpkin seed chyme can be dissolved more thoroughly, thereby ensuring that the protein purity of the prepared pumpkin seed protein is higher, and further ensuring that the constructed sensitized mouse model has better effect.

Further, the weight-to-volume ratio of the pumpkin seed degreasing powder to the leaching buffer solution is 1:8.

in practical application, the leaching buffer comprises 50mmol/L Tris-HCl,200mmol/L NaCl,0.1mmol/L EDTA and 1mmol/L DTT, and the pH value of the buffer is 7.4. Specifically, the isoelectric point value of the pumpkin seed protein is about 5, the albumin accounts for 12%, the globulin accounts for 26%, the prolamine accounts for 8%, the gluten accounts for 54%, and the pumpkin seed protein is mainly composed of two subunits with molecular weights of 20kDa and 35 kDa. Because the content of the glutelin in the pumpkin seed protein is highest, the pumpkin seed protein has poor solubility, and in order to improve the solubility of the pumpkin seed protein, 50mmol/L Tris-HCl leaching buffer solution with the pH value of 7.5 is adopted in the embodiment of the application, so that isoelectric point precipitation of the pumpkin seed protein is avoided. In addition, 200mmol/L NaCl,0.1mmol/L EDTA and 1mmol/L DTT are added into the leaching buffer solution, and the high-concentration NaCl can compete with protein to bind ions in water, so that the dissolution of the protein is promoted; EDTA is used as a cation on the surface of the protein which can be complexed by the polybasic weak acid, so that the pH value of the system is stabilized while the solubility of the protein is improved; DTT can reduce disulfide bonds in secondary and tertiary four-level structures of proteins, break spherical molecular structures of the proteins, and the stretched protein structures are easier to combine with ions in a buffer solution, so that the solubility of the proteins is improved, and the extraction rate of pumpkin seed proteins is increased.

Specifically, the protein solution is dialyzed and then is subjected to vacuum freeze drying to obtain freeze-dried protein powder, which specifically comprises the following steps:

dialyzing the protein solution by using a dialysis bag with a diameter of 16mm and a molecular weight cutoff of 7 kDa; and (3) carrying out vacuum freeze drying on the dialyzed protein liquid to obtain freeze-dried protein powder. In practical application, the dialysis bag with the diameter of 16mm and the cut-off molecular weight of 7kDa is selected for dialyzing the protein liquid, so that salt ions in the protein liquid can be removed, and the pumpkin seed protein can be prevented from being dialyzed into external dialyzate.

In practical application, the freeze-dried protein powder is redissolved in distilled water to obtain freeze-dried protein solution, which specifically comprises the following steps:

in mg/mL, the freeze-dried protein powder and distilled water are mixed according to the following ratio of 1: re-dissolving in the weight-volume ratio of 0.22 to obtain freeze-dried protein solution.

Further, the freeze-dried protein solution is mixed and emulsified with an aluminum hydroxide adjuvant to obtain a sensitization solution, which specifically comprises:

mixing the freeze-dried protein solution with aluminum hydroxide adjuvant according to the following weight ratio of 10:1, and mixing and emulsifying the mixture according to the volume ratio to obtain the sensitization liquid. In practical application, the concentration of the pumpkin seed protein in the sensitization liquid is 4.60mg/mL.

The technical scheme of the application will be further elaborated in the following in conjunction with specific embodiments.

The following is the selection of the formulation of the leaching buffer

1. Purpose(s)

In order to construct a pumpkin seed protein sensitized animal model, a pumpkin seed protein solution with high concentration and no toxicity needs to be prepared. Gluten accounts for about 54% of the pumpkin seed protein, resulting in poor solubility of the pumpkin seed protein. In this embodiment, six leaching buffers with different formulations are mainly used to leach the sensitized proteins in the pumpkin seeds, and the concentration of the pumpkin seed proteins leached by the six leaching buffers with different formulations is compared to select the leaching buffer most suitable for leaching the pumpkin seed proteins.

2. Materials and reagents

The materials and reagents used in this example are commercially available. Specifically, the materials and reagents used in the present experiment were purchased through the following channels.

Pumpkin seed kernel Shaanxi mountain Yanta region Gaoxin Liangye Guangxi Wan Runfu Jiang supermarket;

new urban areas of the western An city of Shaanxi, acetone, yichen, instruments and equipment supply stations;

new urban area of Xishan city of Shaanxi, diethyl ether, yichen, instrument and equipment supply station;

ethylenediamine tetraacetic acid (Ethylene Diamine Tetraacetic Acid, EDTA) beijing complete gold biology company;

sodium dodecyl sulfate (Sodium dodecyl sulfate, SDS) beijing complete gold biology company;

dithiothreitol (DL-Dithiothreitol, DTT) beijing full gold biology company;

Tris-HCl, pH7.5, beijing Soy Bao Co;

new urban areas of the western An city of NaCl Shaanxi are the supply stations of instruments and equipment;

bradford protein concentration determination kit Beijing Soy Bao Co.

3. Main instrument

GL-20B high-speed refrigerated centrifuge was shipped to the Angting laboratory instrumentation plant;

s21-1, shanghai Sele instruments and factories with constant-temperature magnetic stirrers;

AR2140 electronic balance Metler-Tolyduo instruments Co., ltd;

multiskan FC microplate reader, thermo, inc. USA;

2500A mill on the sea ampere pavilion laboratory instrument factory;

TF-JH fume hood Beijing tai Ji ao Fei laboratory equipment Co., ltd;

LGJ-10 freeze dryer beijing four-ring scientific instrumentation factory.

4. Method of operation

1) Degreasing pumpkin seeds

(1) Taking pumpkin seed kernels and liquid nitrogen according to the ratio of 2:1 in a 2500A pulverizer to obtain pumpkin seed emulsion;

(2) firstly, weighing 100g of pumpkin seed emulsion by using an AR2140 electronic balance, then mixing 100g of pumpkin seed emulsion with 1000mL of acetone to obtain a primary mixed solution, magnetically stirring and degreasing the primary mixed solution at 4 ℃ for 2 hours by using an S21-1 constant temperature magnetic stirrer, and finally centrifuging the primary mixed solution for 20 minutes by using a GL-20B high-speed refrigerated centrifuge at 8000rpm to obtain primary precipitate;

(3) first, the primary precipitation and diethyl ether are mixed according to the following formula 1:10, mixing the materials in a weight-volume ratio to obtain a secondary mixed solution, magnetically stirring the secondary mixed solution at 4 ℃ for degreasing for 2 hours by using an S21-1 constant temperature magnetic stirrer, and centrifuging the secondary mixed solution for 20 minutes by using a GL-20B high-speed refrigerated centrifuge at 8000rpm to obtain secondary precipitation;

(4) firstly, secondary precipitation and acetone are mixed according to the following ratio of 1:10, mixing the materials in a weight-volume ratio to obtain a tertiary mixed solution, magnetically stirring the tertiary mixed solution at 4 ℃ for degreasing for 2 hours by using an S21-1 constant temperature magnetic stirrer, and finally centrifuging the tertiary mixed solution for 20 minutes by using a GL-20B high-speed refrigerated centrifuge at 8000rpm to obtain tertiary sediment;

(5) three precipitations were first combined with diethyl ether according to 1: mixing the four mixed solutions according to the weight-volume ratio of 10 to obtain four mixed solutions, magnetically stirring and degreasing the four mixed solutions at 4 ℃ for 2 hours by using an S21-1 constant temperature magnetic stirrer, and centrifuging the four mixed solutions for 20 minutes by using a GL-20B high-speed refrigerated centrifuge at 8000rpm to obtain four precipitates;

(6) and (5) airing the four times of precipitation in a TF-JH fume hood to obtain the pumpkin seed degreasing powder.

2) Preparation of leaching buffer

Six leaching buffers were formulated separately for use in subsequent experiments according to the formulations and pH values shown in table 1 prior to the leaching of the pumpkin seed proteins.

TABLE 1 Leaching buffer formulation

Numbering device	Formulation of	pH
			1	50mmol/L Tris-HCl，200mmol/L NaCl，0.1mmol/L EDTA，1mmol/L DTT	6.5
2	50mmol/L Tris-HCl，200mmol/L NaCl，0.1mmol/L EDTA，10mmol/LSDS，1mmol/L DTT	7.4
			3	50mmol/L Tris-HCl，100mmol/L NaCl，0.1mmol/L EDTA，1mmol/L DTT	7.4
4	50mmol/L Tris-HCl，150mmol/L NaCl，0.1mmol/L EDTA，1mmol/L DTT	7.4
			5	50mmol/L Tris-HCl，200mmol/L NaCl，0.1mmol/L EDTA，1mmol/L DTT	7.4
6	50mmol/L Tris-HCl，200mmol/L NaCl，0.1mmol/L EDTA，1mmol/L DTT	8.0

3) Pumpkin seed protein extraction

The pumpkin seed degreasing powder and six leaching buffers are mixed according to the mass volume ratio of 1:8, respectively magnetically stirring the six mixed liquids at 4 ℃ for 24 hours by using an S21-1 constant temperature magnetic stirrer to obtain six protein thick slurries, respectively centrifuging the six protein thick slurries at 15000r/min for 15 minutes at 4 ℃ by using a GL-20B high-speed refrigerated centrifuge, and finally respectively collecting supernatant liquid of the six protein thick slurries after centrifugation.

4) Protein concentration detection

Protein concentrations in the supernatants of the six protein concentrates were each detected using Bradford protein concentration assay kit.

After the protein concentration measurement is completed, BSA (bovine serum albumin) is used as a standard protein, a protein concentration standard curve is prepared, coomassie brilliant blue G250 is used for dyeing the protein in the leaching buffer solution, the absorption wavelength of a Multiskan FC enzyme-labeled instrument is adjusted to 595nm, the absorbance of each leaching buffer solution is measured, and finally the supernatant with the highest protein content is selected for freeze-drying, so that the pumpkin seed protein freeze-dried powder is obtained.

5. Experimental results

The pumpkin seed protein concentrations in the six leaching buffers are respectively as follows: no. 1 is 3.39.+ -. 0.58mg/mL, no. 2.58.+ -. 0.26mg/mL, no. 3.55.+ -. 0.71mg/mL, no. 4.15.+ -. 0.21mg/mL, no. 5.52.+ -. 0.37mg/mL, no. 6 is 4.51.+ -. 0.47mg/mL (see Table 2).

TABLE 2 pumpkin seed protein concentration table in six leaching buffers

6. Conclusion of the experiment

Among the six leaching buffers, the highest concentration of pumpkin seed protein in the No. 5 leaching buffer is 5.52+/-0.37 mg/mL. As can be seen from table 2, the formulation of the No. 1 leaching buffer was the same as that of the No. 5 leaching buffer, but since the pH of the No. 1 leaching buffer was 6.5, which is closer to the isoelectric point of the pumpkin seed protein, the protein solubility of the No. 1 leaching buffer was inferior to that of the No. 5 leaching buffer. Sodium dodecyl sulfate (Sodium dodecyl sulfate, SDS) is added in the No. 2 leaching buffer solution, and because the dodecyl part in the SDS is of a hydrophobic structure and the sodium sulfonate part is of a hydrophilic structure, the hydrophobic structure of the SDS can be inserted into the hydrophobic structure of the protein to break down the hydrophobic acting force of the protein, so that the three-level and four-level structure of the protein is broken down, and the addition of the SDS can increase the solubility of the protein, but can also cause partial protein denaturation, so that the sensitization activity of the pumpkin seed protein is damaged. The concentration of NaCl in the No. 3, no. 4 and No. 5 leaching buffers was increased stepwise, and correspondingly, the concentration of protein in the leaching buffer was also increased stepwise, and the concentration of protein was highest in this experiment when the concentration of NaCl was 200mmol/L. However, if the concentration of NaCl is too high, protein denaturation is also caused, so that the concentration of NaCl in this experiment is set to a range of 100mmol/L to 200mmol/L. In the leaching buffer No. 6, the pH value is 8.0, but the protein concentration is reduced compared with that of No. 5. In summary, the application selects No. 5 leaching buffer to leach the protein in the pumpkin seeds.

The following is a comparison of the effects of a sensitized mouse model constructed from short-term sensitization and low-dose long-term sensitization

1. Purpose(s)

Short-term sensitization is a common sensitization mode used in the construction of a sensitization mouse model, and the aim of the research in the section is to determine an animal model which can comprehensively represent typical characteristics of human pumpkin seed protein allergy and provide a large amount of allergic serum to meet the research requirement by comparing the effects of short-term sensitization and low-dose long-term sensitization construction models.

2. Test object and material

1) Animal preparation

Female BALB/c mice of 4 weeks old 24 (SPF grade), weighing 8g-12g, were fed by Chengdu laboratory animal Co., ltd., free diet and drinking water in the North-West university animal house. Feeding environment: the temperature is 22-25 ℃; humidity: 40% -60%.

2) Materials and reagents

The materials and reagents used in this example are commercially available. Specifically, the materials and reagents used in the present experiment were purchased through the following channels.

Aluminum hydroxide adjuvant (Imject Alum Adjuvant) sameifeishi technologies limited;

Tris-HCl Beijing Soy Bao technology Co., ltd at pH 7.5;

immunoglobulin E (IgE) detection kit Wuhan cloud clone technologies Co., ltd;

histamine (HA) detection kit, wuhan cloud clone technologies, inc.;

IL-4 detection kit Shanghai Xinle Biotechnology Co., ltd;

IL-13 detection kit Shanghai Xinle Biotechnology Co., ltd;

4% paraformaldehyde solution is supplied to the instrument and equipment supply station in New urban area of western An, shaanxi.

3) Main instrument

Multiskan FC microplate reader, thermo, inc. USA;

MX-F vortex Mixer Sirocco instruments, inc.

4) Grouping modeling

24 mice were acclimatized for 3 weeks and then randomized into solvent control (solvent control), adjuvant control (adjuvant control), short-term sensitization (short-term) and low-dose long-term sensitization (long-term), 6 each. The test flow is shown in fig. 1, fig. 2, fig. 3 and fig. 4.

Sensitization: re-dissolving pumpkin seed protein freeze-dried powder in distilled water to obtain freeze-dried protein dissolving solution, wherein the volume ratio of the freeze-dried protein dissolving solution is 10:1 adding an aluminum hydroxide adjuvant (namely, adding 1mL of the aluminum hydroxide adjuvant into 10mL of freeze-dried protein solution), and repeatedly oscillating the solution to uniformly emulsify the solution to prepare the sensitized liquid with the protein concentration of 4.60mg/mL.

Short-term sensitization group: mice were intraperitoneally injected with 200 μl of sensitization solution at days 21, 28, 35, 42;

low dose long-term sensitization group: mice were intraperitoneally injected with 150 μl of sensitization solution on days 21, 28, 35, 42, 49, 56;

solvent control group: mice were intraperitoneally injected with 200. Mu.L of Tris-HCl buffer at days 21, 28, 35, 42, 49, 56. Specifically, the formulation of Tris-HCl buffer was 50mmol/L Tris-HCl,200mmol/L NaCl,0.1mmol/L EDTA and 1mmol/L DTT, and the pH of Tris-HCl buffer was 7.4.

Adjuvant control group: according to the volume ratio of 10:1 mixing Tris-HCl buffer and aluminium hydroxide adjuvant to form injection adjuvant, 200. Mu.L of the adjuvant was intraperitoneally injected into mice on days 21, 28, 35, 42, 49, 56, respectively.

Excitation: the mice of the low-dose long-term sensitization group are stimulated on the 62 th day, 200mg of pumpkin seed freeze-dried protein powder is mixed with 10mL of distilled water, and the mixture is fully stirred to prepare protein liquid, and then each mouse is filled with 500 mu L of protein liquid. Short-term sensitized group mice were challenged on day 49 with 500 μl of protein fluid per lavage. Both the solvent control and the adjuvant control were challenged on day 62 and each mouse was perfused with 500 μ LTris-HCl buffer.

5) Specimen collection

Serum

Short-term sensitized group mice were subjected to eyeball removal on day 49 to remove blood. The specific operation is as follows: the beard of the mice was cut off, one side eyeball was removed, blood was collected with a 1.5mL centrifuge tube, and the mice were sacrificed by cervical removal. The blood was left at room temperature for 2h, then centrifuged at 3000r/min at 4℃for 10min, after centrifugation the supernatant serum was taken and placed in a further EP tube and stored at-80 ℃.

The mice in the low-dose long-term sensitized group were subjected to intracanthus venous blood removal on days 45, 49, 52, 56 and 59, and eyeball blood removal on day 62. The process of obtaining blood from the inner canthus vein is as follows: the operator uses the thumb, index finger and middle finger of the left hand to fix the head of the mouse, then squeezes the head of the mouse to enable the eyeballs of the mouse to protrude, then the right hand holds the capillary tube to penetrate from the inner canthus and form a 45-degree included angle with the mouse surface in a rotating mode, finally the body of the mouse is fixed, the limbs are relaxed, blood is adjusted to smoothly drop out from the rear of the capillary tube, a 1.5mL centrifuge tube is used for collecting the blood, the capillary tube is gently pulled out after blood taking, and a dry cotton ball is used for pressing the eyeorbit of the mouse to stop bleeding. Standing at room temperature for 2 hr, centrifuging at 3000r/min and 4deg.C for 10min, collecting upper serum, and storing at-80deg.C.

Solvent control and adjuvant control mice were subjected to eye-picking on day 62 for blood collection, with specific procedures consistent with short-term sensitized mice.

Colon tissue: the mice were dissected, 2cm colon tissue was taken and fixed in 4% paraformaldehyde fixing solution for pathological section production.

3. Research method

1) Allergic apparent symptom score

The clinical symptoms of mice were classified into 6 classes in this experiment: 0= "asymptomatic"; 1= "grab nose and head"; 2= "activity decrease or respiratory rate increase"; 3= "dyspnea, redness around eyes and/or mouth, hair loss"; 4= "tic or immobility after oral stimulation"; 5= "death". Clinical scores 4 and 5 are classified as severe allergic symptoms, 2 and 3 are classified as moderate allergic symptoms, and 1 is classified as mild allergic symptoms. Mice developed allergic symptoms after 0.5h of gastric lavage challenge and were scored according to allergic symptoms.

2) Detection of the content of various factors in serum

The total IgE, pumpkin seed protein specific IgE, histamine, IL-4 and IL-13 content in the mouse serum was measured according to the instructions for the immunoglobulin E (IgE) detection kit, the instructions for the Histamine (HA) detection kit, the instructions for the IL-4 detection kit and the instructions for the IL-13 detection kit, respectively.

3) Colon histopathological staining observation of mice

After dissecting mice and taking intestinal tissues for soaking, the mice are sent to Shaanxi Eyew Biotechnology service Co.Ltd for paraffin embedding, paraffin sectioning and hematoxylin-eosin staining.

4) Data statistics

Data are expressed as "mean ± standard deviation", mean difference significance test between samples is analyzed by ANOVA, and if there is a significant difference, the LSD (mean square error) and Benferroni (mean square error) methods are used for pairwise comparison. When the variance is irregular, a rank sum test is used and SPSS20.0 statistical software is used for data analysis. P <0.05 is considered to be significant, and significant differences between the data are noted with lower case letters a, b, c, etc.

4. Results of the study

1) Comparison of allergic signs in mice of groups

As shown in FIG. 5, the average body weight of the mice in the solvent control group was about 28.06.+ -. 3.01g, the average body weight of the mice in the adjuvant control group was about 25.21.+ -. 2.09g, the average body weight of the mice in the short-term sensitized group was about 17.89.+ -. 3.32g, and the average body weight of the mice in the low-dose long-term sensitized group was about 18.20.+ -. 1.52g. From the above data, the body weight of the mice in the short-term sensitized group and the mice in the low-dose long-term sensitized group were significantly lower than those in the solvent control group and the adjuvant control group, which indicates that both the mice in the short-term sensitized group and the mice in the low-dose long-term sensitized group were allergic. This is because food allergy easily causes inflammation at various sites in the body and also often causes impaired gastrointestinal function, so that the body weight of allergic mice tends to be reduced as compared with normal mice.

As shown in fig. 6 and 7, the spleen index and thymus index of the solvent control mice were significantly lower than those of the short-term sensitized mice and the low-dose long-term sensitized mice. The higher spleen index and thymus index indicate that the higher the intensity of immune response in the organism, the higher the spleen/thymus index of the mice indicate that immune organs and immune cells are in an active state, and the higher the probability of excessive immune response (i.e. allergy) occurs, thereby indicating that allergy occurs in both short-term sensitized mice and low-dose long-term sensitized mice.

As shown in fig. 8, the short-term sensitized group mice and the low-dose long-term sensitized group mice showed a significantly reduced body temperature compared to the solvent control group and the adjuvant control group, because the blood supply to the body temperature center was reduced due to the vasodilation in the body of the sensitized mice, and the body temperature was reduced as well, thereby indicating that both the short-term sensitized group mice and the low-dose long-term sensitized group mice were sensitized.

As shown in fig. 9, mice in the solvent control group and the adjuvant control group had lower allergic symptoms scores, indicating that neither group had obvious allergic symptoms. The mice in the short-term sensitization group and the mice in the low-dose long-term sensitization group have higher allergic symptoms, and the mice in the low-dose long-term sensitization group have higher allergic symptoms than the mice in the short-term sensitization group, which means that the allergic symptoms generated by the mice are more obvious due to the low-dose long-term sensitization than the short-term sensitization.

2) Determination of total IgE and pumpkin seed protein specific IgE content in mouse serum

As shown in FIG. 10, the total IgE content in the mouse serum of the solvent control group was 9.08.+ -. 0.73ng/mL, the total IgE content in the mouse serum of the adjuvant control group was 8.98.+ -. 1.23ng/mL, the total IgE content in the mouse serum of the short-term sensitized group was 26.08.+ -. 2.53ng/mL, and the total IgE content in the mouse serum of the low-dose long-term sensitized group was 28.66.+ -. 0.59ng/mL. From the above data, the total IgE content in the serum of mice in the short-term sensitized group and the low-dose long-term sensitized group was significantly higher than that in the solvent control group and the adjuvant control group. The higher total IgE content in the serum of mice in the low-dose long-term sensitized group compared to the short-term sensitized group suggests that low-dose long-term sensitization can produce more IgE in the serum of mice.

As shown in fig. 11, the pumpkin seed protein specific IgE content in the serum of the mice was relatively similar in the solvent control group and the adjuvant control group, which was 56.36% lower than that in the short-term sensitized group, 52.08% lower than that in the low-dose long-term sensitized group, and there was no significant difference in the pumpkin seed protein specific IgE content in the short-term sensitized group and the low-dose long-term sensitized group. It was demonstrated that both the short-term sensitized group and the low-dose long-term sensitized group elicited an immune response compared to the solvent control group and the adjuvant control group.

3) Determination of histamine content in mouse serum

As shown in FIG. 12, the histamine content in the serum of the mice of the solvent control group was 10.06.+ -. 1.04ng/mL, the histamine content in the serum of the mice of the adjuvant control group was 9.97.+ -. 3.91ng/mL, the histamine content in the serum of the mice of the short-term sensitized group was 40.15.+ -. 5.25ng/mL, the histamine content in the serum of the mice of the low-dose long-term sensitized group was 39.44.+ -. 3.97ng/mL, and the histamine contents in the serum of the mice of the solvent control group and the serum of the mice of the adjuvant control group were relatively close. From the above data, the histamine content in the serum of mice in the short-term sensitized and low-dose long-term sensitized groups was significantly higher than that in the solvent control group and the adjuvant control group.

For low dose long term sensitized group mice, the experiment was also performed with blood collection on days 45, 49, 52, 56, 59, 62 of the experiment and the total IgE and histamine content in the blood was examined. As shown in fig. 13, the total IgE and histamine were at high levels in the serum obtained by 6 blood collection, and the total IgE and histamine content in the serum obtained by 62 blood collection reached the highest value in 6 samples, which indicates that the mice developed severe allergic reactions at the time of large-dose gastric lavage with pumpkin seed protein.

4) Determination of Th2 type cytokine content in mouse serum

As shown in fig. 14 and 15, the levels of Th 2-type cytokine (IL-4, IL-13) in serum of mice in both the short-term sensitization group and the low-dose long-term sensitization group were significantly increased as compared to the solvent control group and the adjuvant control group, but there was no significant difference between the two modes of short-term sensitization and low-dose long-term sensitization, which indicates that the mice were able to develop significant immune responses in both the short-term sensitization and the low-dose long-term sensitization modes.

5) Histopathological observations

The HE stained sections were visualized under a mirror, and it was seen that the intestinal mucosa of the mice was intact in structure, the epithelial cells were closely aligned, the intestinal glands of the lamina propria were abundant, the goblet cells were more, and there were no obvious signs of inflammation in the solvent control group and the adjuvant control group (fig. 16a, fig. 16 b). The colon tissue of mice in the short-term sensitized group and the low-dose long-term sensitized group showed a large number of disrupted nuclei and inflammatory infiltrates (fig. 16c, fig. 16 d).

6) Comparison of blood collection and serum volumes of mice of each group

Short-term sensitization groups were extirpated with blood taken from the eyeballs on day 49, with an average blood sampling per mouse of about 500 μl and a serum volume of about 200 μl. The low-dose long-term sensitization group was subjected to inner canthus vein removal on days 45, 49, 52, 56 and 59, eyeball removal on day 62, total blood collection times of 6 times, average blood collection amount per mouse was about 700. Mu.L, and serum volume was about 320. Mu.L. The solvent control and adjuvant control were extirpated to collect blood on day 62, with an average blood collection per mouse of about 500 μl and a serum volume of about 200 μl. From the above data, it can be seen that the blood collection amount and serum amount of the low-dose long-term sensitized group are relatively high compared to the short-term sensitized group.

5. Conclusion of the experiment

The construction of the pumpkin seed sensitized mouse model mainly comprises two parts: sensitization and sensitization, wherein the sensitization degree of mice is influenced by a buffer system in which the sensitization source is positioned and the sensitization mode of the sensitization source, and in the research of the part, the effect of constructing a model for short-term sensitization and low-dose long-term sensitization mode is compared by grouping and modeling the mice. According to experimental results, compared with a short-term sensitization group, the total IgE content in the serum of the mice in the low-dose long-term sensitization group is higher, allergic symptoms are more obvious, the blood sampling times are more, and the available sensitization serum amount is more. Therefore, a pumpkin seed protein sensitized mouse model is successfully constructed by a mode of low-dose long-term sensitization, namely, a mode of carrying out intraperitoneal injection of sensitization liquid and gastric lavage of a mouse for a small number of times, and the anaphylaxis symptom of the mouse model is obvious, so that the typical characteristic of human pumpkin seed protein allergy can be well simulated. Meanwhile, the sensitization state of the mice is prolonged through low-dose long-term sensitization in the model construction process, so that the blood sampling times of the mice are increased, the blood sampling amount is increased, the sensitization serum amount is also increased, and further technical support and material basis are provided for further researching the sensitization mechanism of pumpkin seed proteins, so that the requirements of subsequent researches are met.

In this specification, each embodiment is described in a progressive manner, and the same or similar parts of each embodiment are referred to each other, and each embodiment is mainly described as a difference from other embodiments.

The above embodiments are only for illustrating the technical solution of the present application, and not for limiting the present application; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced with equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims (5)

1. The construction method of the pumpkin seed protein sensitized mouse model is characterized by comprising the following steps of:

repeatedly leaching the pumpkin seed degreasing powder with leaching buffer solution for multiple times to obtain protein solution;

dialyzing the protein liquid, and performing vacuum freeze drying to obtain freeze-dried protein powder;

re-dissolving the freeze-dried protein powder in distilled water to obtain freeze-dried protein solution, which comprises the following steps:

the freeze-dried protein powder and distilled water are mixed according to the ratio of 1: re-dissolving in a weight-volume ratio of 0.22 to obtain freeze-dried protein solution;

mixing and emulsifying the freeze-dried protein solution and an aluminum hydroxide adjuvant to obtain a sensitization solution, wherein the sensitization solution specifically comprises the following components:

mixing the freeze-dried protein solution with an aluminum hydroxide adjuvant according to a ratio of 10:1, mixing and emulsifying the mixture in a volume ratio to obtain a sensitization liquid;

a plurality of 3-4 week old BALB/c mice are adaptively fed for 3 weeks, and 150 mu L of the sensitization liquid is respectively injected into each of the mice from the abdominal cavity on days 21, 28, 35, 42, 49 and 56;

each mouse was perfused with 8mg-10mg of pumpkin seed protein buffer on day 62.

2. The method for constructing a pumpkin seed protein sensitized mouse model according to claim 1, wherein before the pumpkin seed defatted powder is repeatedly leached with the leaching buffer solution for a plurality of times to obtain a protein solution, the method further comprises:

taking pumpkin seed kernels and liquid nitrogen according to the ratio of 2:1, grinding in a grinder to obtain pumpkin seed emulsion;

mixing the pumpkin seed emulsion with a fat solvent for multiple times, centrifuging and precipitating to obtain a pumpkin seed degreasing precipitate;

and (3) airing the pumpkin seed degreasing precipitate in a fume hood to obtain the pumpkin seed degreasing powder.

3. The method for constructing a pumpkin seed protein sensitized mouse model according to claim 1, wherein the weight-to-volume ratio of the pumpkin seed degreasing powder to the leaching buffer is 1:8.

4. the method for constructing a pumpkin seed protein sensitized mouse model according to claim 1, wherein the leaching buffer comprises 50mmol/L Tris-HCl,200mmol/L NaCl,0.1mmol/L EDTA and 1mmol/L DTT, and the pH value of the buffer is 7.4.

5. The method for constructing a pumpkin seed protein sensitized mouse model according to claim 1, wherein the freeze-dried protein powder is obtained by vacuum freeze-drying after the protein liquid is dialyzed, and specifically comprises the following steps:

dialyzing the protein solution by using a dialysis bag with a diameter of 16mm and a molecular weight cut-off of 7 kDa;

and carrying out vacuum freeze drying on the dialyzed protein liquid to obtain the freeze-dried protein powder.