Soybean sensitization protein quantitative detection system based on western blot technology and construction method

Abstract

The invention belongs to the technical field of immunodetection, and particularly discloses a soybean allergenic protein quantitative detection system based on a western blot technology and a construction method, and the construction method comprises the following steps: constructing a standard curve for detecting soybean allergenic protein based on the western blot technology; performing a repetitive verification test; adding a standard recovery rate verification test; and extracting total protein in a to-be-detected soybean product sample, performing quantitative analysis according to the standard curve, and calculating the content of the soybean allergenic protein and the subunit or peptide chain thereof in the sample. According to the detection system and the construction method provided by the invention, the total amount of the soybean allergenic protein and the number of subunits or peptide chains thereof can be detected at the same time, and the detection precision and the operation efficiency of the soybean allergenic protein are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and particularly relates to a quantitative detection system for soybean allergenic proteins based on western blotting technology and a construction method thereof. Background Art

[0002] Soybean allergenic proteins, mainly glycinin and β-conglycinin, are proteins present in soybeans and their products that can cause allergic reactions in humans and animals. Glycinin is a hexamer composed of six subunits, each subunit containing an acidic peptide chain and a basic peptide chain; β-conglycinin is a trimeric globulin composed of α, α' and β subunits. Their complex structures endow them with specific anti-digestion characteristics and allergenicity, and each subunit and peptide chain has allergenicity.

[0003] Currently, enzyme-linked immunosorbent assay (ELISA) is mainly used to detect the contents of glycinin and β-conglycinin in samples, and quantitative analysis is achieved by preparing specific antibodies. However, ELISA technology has limitations. It cannot accurately determine the specific quantities of each subunit or peptide chain, and allergenicity is often related to specific subunits or peptide chains. Only detecting the total amount limits the in-depth understanding and precise prevention and control of allergic reactions. In addition, ELISA is cumbersome to operate, affected by various experimental conditions, and the reproducibility of results is limited, which is not conducive to rapid and accurate detection in large-scale production and quality control. Therefore, it is very necessary to develop a new technology that can simultaneously detect the total amounts of glycinin and β-conglycinin and the quantities of their subunits or peptide chains. Summary of the Invention

[0004] The present invention aims to provide a quantitative detection system for soybean allergenic proteins based on western blotting technology and a construction method thereof. This system can simultaneously detect the total amounts of glycinin and β-conglycinin and the quantities of their subunits or peptide chains, significantly improving the detection accuracy and operation efficiency of soybean allergenic proteins.

[0005] To solve the above technical problems, the technical scheme adopted by the present invention is as follows:

[0006] A construction method for a quantitative detection system for soybean allergenic proteins based on western blotting technology includes the following steps:

[0007] S1. Construct a standard curve for detecting soybean allergenic proteins based on western blotting technology;

[0008] S2. Reproducibility verification test;

[0009] S3. Spike recovery verification test;

[0010] S4. Extract the total protein from the sample of the soy product to be tested, perform quantitative analysis based on the standard curve, and calculate the content of soy allergenic protein and its subunits or peptide chains in the sample.

[0011] Further, in S1, the method for constructing the standard curve for detecting soy allergenic protein based on Western blotting technology is as follows:

[0012] A1. Isolate and obtain purified soy allergenic protein;

[0013] A2. Use the soy allergenic protein obtained in A1 as an immunogen to immunize rabbits, and obtain polyclonal antibodies through affinity chromatography of the immune serum.

[0014] A3. Denature the purified soy allergenic protein obtained in A1 to obtain denatured-soy allergenic protein;

[0015] A4. Perform SDS-polyacrylamide gel electrophoresis on the denatured-soy allergenic protein obtained in A3 to obtain a denatured-soy allergenic protein gel;

[0016] A5. Transfer the denatured-soy allergenic protein gel obtained in A4 to a PVDF membrane to obtain a denatured-soy allergenic protein transfer membrane;

[0017] A6. After blocking the denatured-soy allergenic protein transfer membrane obtained in A5 with a blocking buffer, add the polyclonal antibodies obtained in A2, incubate overnight, wash the membrane, then add horseradish peroxidase-labeled anti-rabbit IgG antibody for incubation, and add a chemiluminescent substrate for color development.

[0018] Use a gel imaging system to collect the membrane image after color development, and perform optical density analysis on each band using analysis software to establish a standard curve for detecting soy allergenic protein based on Western blotting technology.

[0019] Further, in S1, the soy allergenic protein is one or both of glycinin or β-conglycinin.

[0020] Further, in A1, the method for isolating purified soy allergenic protein includes the following steps:

[0021] B1. Select intact soybeans, crush them, and sieve them to obtain soybean powder;

[0022] B2. Add 4°C cold acetone to the soybean powder obtained in B1, stir at 500 rpm at room temperature for 1 h, let it stand for 30 min, discard the supernatant, repeat the above process until the supernatant is clear, discard the supernatant, and air-dry the precipitate naturally to obtain defatted soybean powder;

[0023] B3. Crude extraction of soybean allergenic protein: Add extraction buffer to the defatted soybean powder obtained in B2, stir for extraction, then centrifuge and discard the precipitate. Adjust the pH, let it stand and centrifuge again, collect precipitate A. Adjust the pH value of the supernatant, stir and let it stand, then collect precipitate B. The obtained precipitate A and precipitate B are the crude extracted soybean allergenic protein;

[0024] B4. Dissolve the crude extracted soybean allergenic protein obtained in B3 in PBS buffer, heat it in a water bath to 25°C, add solid ammonium sulfate, dissolve it thoroughly, collect the supernatant, heat it in a water bath to 25°C, and continue to add solid ammonium sulfate, then collect the precipitate;

[0025] B5. Add PBS buffer to dissolve the precipitate obtained in B4, transfer it to a dialysis bag, dialyze at 4°C for 48 h, collect the dialysate, and freeze-dry to obtain purified soybean allergenic protein.

[0026] Further, in S2, the repeatability verification test includes the following steps:

[0027] C1. Select soybean product samples of the same batch, and repeatedly measure the content of soybean allergenic protein and its subunits or peptide chains for multiple times;

[0028] C2. Calculate the average value and standard deviation of the measurement results;

[0029] C3. Calculate the relative standard deviation based on the standard deviation to evaluate the repeatability and stability of the detection method.

[0030] Further, in S3, the spike recovery verification test includes the following steps:

[0031] D1. Add a known amount of purified soybean allergenic protein standard to the soybean product sample to form a spiked sample;

[0032] D2. Measure the content of soybean allergenic protein and its subunits or peptide chains in the spiked sample;

[0033] D3. Calculate the recovery rate of soybean allergenic protein and its subunits or peptide chains in the spiked sample.

[0034] Further, in S4, when extracting the total protein in the soybean product sample to be tested and performing quantitative analysis based on the standard curve to calculate the content of soybean allergenic protein and its subunits or peptide chains, the calculation formula is:

[0035]

[0036] Among them, C is the content of soybean allergenic protein and its subunits or peptide chains in the sample, with the unit of mg / g; m is the amount of soybean allergenic protein and its subunits or peptide chains calculated using the standard curve equation, with the unit of ng; M is the mass of the weighed sample, with the unit of g; V is the total volume of the supernatant obtained by centrifuging the total protein extracted from the sample with the extraction buffer, with the unit of mL; v is the sample loading volume during electrophoresis, with the unit of μL.

[0037] Application of the construction method as described above in generating a quantitative detection system for soybean allergenic protein based on Western blotting technology.

[0038] Compared with the prior art, the present invention has the following advantages and technical effects:

[0039] The present invention discloses a quantitative detection system and construction method for soybean allergenic protein based on Western blotting technology. The detection accuracy and resolution have been greatly improved. It can not only measure the total amount of glycinin or β-conglycinin, but also accurately distinguish the content of each subunit or peptide chain. With the help of high-resolution gel electrophoresis separation, Western blotting technology and specific antibody detection, it can accurately analyze the allergenic protein, providing data support for the research of allergenic mechanisms, etc. And the anti-interference ability is significantly enhanced. By optimizing key steps such as electrophoresis, membrane transfer, and color development, the interference of impurity proteins is reduced, ensuring the reliability of the detection results in complex sample matrices. The operation process is simplified and efficient, meeting the rapid detection requirements for quality control in large-scale production.

[0040] The construction method of the present invention has a wide range of applications. It is not only suitable for laboratory research, but also plays an important role in the food and feed industries. It can be used for quality control of various soybean products, food allergen management, feed formula optimization, etc., providing advanced and reliable technical means for the detection research of soybean allergenic protein, and having important innovative significance and broad application value.

[0041] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0042] Figure 1 Standard curve of glycinin provided for Example 4;

[0043] Figure 2 Standard curve of glycinin A3 subunit provided for Example 4;

[0044] Figure 3 Standard curve of glycinin AS subunit provided for Example 4;

[0045] Figure 4 Standard curve of glycinin BS subunit provided for Example 4;

[0046] Figure 5β-conglycinin standard curve provided for Example 4;

[0047] Figure 6 β-conglycinin α'-subunit standard curve provided for Example 4;

[0048] Figure 7 β-conglycinin α-subunit standard curve provided for Example 4;

[0049] Figure 8 β-conglycinin β-subunit standard curve provided for Example 4. Detailed implementation mode

[0050] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and examples.

[0051] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs.

[0052] In the present invention, unless otherwise specified, other test materials and instrument equipment are all conventional test materials in the art and can be obtained through commercial channels.

[0053] Preparation of buffers used in the examples:

[0054] Extraction buffer: 0.03 mol / L Tris-HCl, containing 10 mmol / L β-mercaptoethanol, pH 8.0;

[0055] TBST buffer: 20 mmol / L Tris, 150 mmol / L NaCl, 0.05% Tween-20, pH 8.0;

[0056] Blocking buffer: TBST containing 5% skim milk powder.

[0057] PBS buffer: pH = 7.4, 2.6 mmol / L KH 2 PO 4 ,32.5 mmol / L K 2 HPO 4 ,

[0058] 0.4 mol / L NaCl, containing 10 mmol / L β-ME;

[0059] Glycine-HCl buffer (0.1 mol / L): Weigh 7.507 g of glycine, dissolve it in 800 mL of deionized water, adjust the pH of the solution to 2.4 with concentrated hydrochloric acid, and make up the volume to 1 L.

[0060] Tris-HCl buffer (1.5 mol / L): Weigh 181.7 g of Tris, dissolve it in 800 mL of deionized water, adjust the pH of the solution to 8.8 with concentrated hydrochloric acid, and make up the volume to 1 L;

[0061] Tris-HCl buffer (1.0 mol / L): Weigh 121.1 g of Tris, dissolve it in 800 mL of deionized water, adjust the pH of the solution to 6.8 with concentrated hydrochloric acid, and make up the volume to 1 L;

[0062] 5× Loading buffer: 250 mmol / L Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol;

[0063] Electrophoresis buffer: 25 mmol / L Tris, 193 mmol / L glycine, 0.1% SDS, pH 8.3;

[0064] Transfer buffer: 25 mmol / L Tris, 193 mmol / L glycine, 20% methanol, pH 8.3.

[0065] Example 1

[0066] A method for extracting purified soy globulin, comprising the following steps:

[0067] B1. Select intact soybeans, crush them, and pass through a 60-mesh sieve to obtain soybean powder;

[0068] B2. Add 500 mL of 4°C cold acetone to 100 g of the soybean powder obtained in B1, seal the cup mouth with a sealing film, place it on a magnetic stirrer, stir at 500 rpm at room temperature for 1 h, let it stand for 30 min, discard the supernatant, add 500 mL of 4°C cold acetone again, repeat the above process, change the liquid twice until the supernatant is clear, discard the supernatant, transfer the precipitate to a fume hood, and naturally air-dry the remaining acetone to obtain defatted soybean powder.

[0069] B3. Weigh 50 g of the defatted soybean powder obtained in B2, add 1000 mL of extraction buffer, place it on a magnetic stirrer and stir at 1000 rpm at room temperature for 1 h, centrifuge at 9000 g at 4°C for 30 min, discard the precipitate, adjust the pH value of the supernatant to 6.4 with 2 mol / L HCl, let it stand overnight at 4°C, centrifuge at 9000 g at 4°C for 20 min, collect the precipitate to obtain crude soy globulin;

[0070] B4. Dissolve the crude soybean globulin obtained in B3 in PBS buffer, heat it in a water bath to 25°C, add solid ammonium sulfate to make the solution reach 51% ammonium sulfate saturation, centrifuge at 4°C and 12,000 g for 20 min, collect the supernatant, heat it in a water bath to 25°C, add solid ammonium sulfate to make the solution reach 66% ammonium sulfate saturation, centrifuge at 4°C and 12,000 g for 20 min, collect the precipitate to obtain soybean globulin.

[0071] The addition amount of solid ammonium sulfate is calculated according to the following formula:

[0072]

[0073] In the formula: g represents the grams of solid ammonium sulfate added to 1 L of solution; S 1 represents the percentage saturation of ammonium sulfate in the original solution; S 2 represents the required percentage saturation of ammonium sulfate.

[0074] B5. Add an appropriate amount of PBS buffer to the soybean globulin obtained in B4 to dissolve it, clamp both ends of the dialysis bag with dialysis clips to avoid liquid leakage, place it in PBS buffer at pH 7.4, stir at 300 rpm on a magnetic stirrer, dialyze at 4°C for 24 h, change the liquid 3 times in the middle. When changing the liquid for the 4th time, change the dialysis liquid to distilled water, continue to dialyze for 24 h, change the liquid 3 times, centrifuge at 4°C and 9,000 g for 10 min, collect the supernatant, put it into a freeze-drying bottle, seal it with a sealing film and place it in a -20°C refrigerator. Wait until the liquid is completely frozen, install the freeze-drying bottle on the freeze-dryer, and freeze-dry at -40°C for 5 h to obtain purified soybean globulin.

[0075] Example 2

[0076] A method for extracting β-conglycinin, comprising the following steps:

[0077] B1. Select intact soybeans, crush them, and pass through a 60-mesh sieve to obtain soybean powder;

[0078] B2. Weigh 100 g of the soybean powder obtained in B1, add 500 mL of 4°C cold acetone, seal the cup mouth with a sealing film, place it on a magnetic stirrer, stir at 500 rpm at room temperature for 1 h, let it stand for 30 min, discard the supernatant, add 500 mL of 4°C cold acetone again, repeat the above process, change the liquid 2 times until the supernatant is clear, discard the supernatant, transfer the precipitate to a fume hood, and naturally air-dry the remaining acetone to obtain defatted soybean powder.

[0079] B3. Add 50 g of the defatted soy flour obtained in B2 to 1000 mL of extraction buffer, place it on a magnetic stirrer and stir at 1000 rpm at room temperature for 1 h. Centrifuge at 9000 g for 30 min at 4°C, discard the precipitate, collect the supernatant, adjust the pH to 6.4 with 2 mol / L HCl, let it stand overnight at 4°C, centrifuge at 9000 g for 20 min at 4°C, collect the supernatant, adjust the pH to 5.5 with 2 mol / L HCl, stir with a magnetic stirrer at 500 rpm for 30 min, centrifuge at 9000 g for 30 min at 4°C, discard the precipitate, dilute it 2-fold with distilled water, adjust the pH of the solution to 4.8 with 2 mol / L HCl, let it stand overnight at 4°C, centrifuge at 9000 g for 30 min at 4°C, collect the precipitate to obtain the crudely extracted β-conglycinin;

[0080] B4. Dissolve the crudely extracted β-conglycinin obtained in B3 in PBS buffer, heat it in a water bath to 25°C, add solid ammonium sulfate to make the solution reach 51% ammonium sulfate saturation, centrifuge at 12000 g for 20 min at 4°C, collect the supernatant, heat it in a water bath to 25°C, add solid ammonium sulfate to make the solution reach 100% ammonium sulfate saturation, centrifuge at 12000 g for 20 min at 4°C, collect the precipitate to obtain β-conglycinin;

[0081] Among them, the calculation formula for the addition amount of solid ammonium sulfate is the same as that in Example 1.

[0082] B5. Add an appropriate amount of PBS buffer to the β-conglycinin obtained in B4 to dissolve it, clamp both ends of the dialysis bag with dialysis clips to avoid liquid leakage, put it into PBS buffer with a pH of 7.4, stir on a magnetic stirrer at 300 rpm, dialyze at 4°C for 24 h, change the liquid 3 times in the middle. When changing the liquid for the 4th time, change the dialysis liquid to distilled water and continue to dialyze for 24 h, change the liquid 3 times, centrifuge at 9000 g for 10 min at 4°C, collect the supernatant, put it into a freeze-drying bottle, seal it with a sealing film and put it into a -20°C refrigerator. Wait until the liquid is completely frozen, install the freeze-drying bottle on the freeze-dryer, freeze-dry at -40°C for 5 h to obtain the purified β-conglycinin.

[0083] Example 3

[0084] Prepare rabbit anti-soybean globulin polyclonal antibody, and the steps are as follows:

[0085] Select the purified soybean globulin prepared in Example 1.

[0086] (1) Use a pipette to aspirate 1 mL of Freund's complete adjuvant (Freund's incomplete adjuvant for booster immunization), accurately weigh 1 mg of the purified soy globulin prepared in Example 1 into a centrifuge tube, add 1 mL of sterile PBS, mix well and then mix with the adjuvant in equal volume at a ratio of 1:1. Place the centrifuge tube on ice and use an emulsifier for sufficient emulsification. When the emulsified liquid does not disperse when dropped into cold water, the emulsification is completed;

[0087] (2) Primary immunization: Select healthy New Zealand white rabbits (body weight 2.5 kg) and perform subcutaneous multi-point injection with a syringe at 0.8 mL / rabbit (400 μg antigen);

[0088] (3) Booster immunization: Perform booster immunization two weeks after the primary immunization, with the same dose as the primary immunization. Then perform booster immunization once every week. After the sixth immunization, the antigen is doubled (800 μg), and a total of eight immunizations are performed.

[0089] (4) Blood collection and detection: One week after each immunization, collect a small amount of blood from the marginal ear vein of the rabbit, separate the serum, and measure the antibody titer. When the antibody titer reaches 1:1000000 or more, perform terminal blood collection one week after the last immunization. Let the collected blood stand for a period of time and then centrifuge (3500 g, 20 min, 4 °C) to separate the serum;

[0090] (5) Using the purified soy globulin obtained in Example 1 as a ligand, prepare an immunoaffinity chromatography column. Use CNBr-activated Sepharose 4B as the activated packing material, couple the antigen to the packing material, dilute the serum obtained in step (4) with PBS buffer, and slowly add it to the equilibrated affinity chromatography column. Wash the column with PBS buffer to wash away the unbound proteins, elute the specifically bound antibodies with glycine-HCl buffer, collect the eluate, and immediately neutralize it with 1 mol / L sodium bicarbonate solution to obtain rabbit anti-soy globulin polyclonal antibody.

[0091] Prepare rabbit anti-β-conglycinin polyclonal antibody, and the preparation method is the same as that for preparing soy globulin polyclonal antibody. The difference is that: select the purified β-conglycinin prepared in Example 2 to obtain rabbit anti-β-conglycinin polyclonal antibody.

[0092] Example 4

[0093] Construct a standard curve for detecting soy globulin based on the Western blotting technique, and the construction method is as follows:

[0094] A1. Select the purified soy globulin prepared in Example 1;

[0095] A2. Select the rabbit anti-soy globulin polyclonal antibody prepared in Example 3;

[0096] A3. Weigh accurately 1.0 mg of the purified soy globulin obtained from A1, dissolve it in 1.0 mL of PBS buffer, vortex until completely dissolved, then add 0.25 mL of 5× loading buffer, and boil for 10 min to obtain denatured soy globulin.

[0097] A4. Perform SDS-polyacrylamide gel electrophoresis on the denatured soy globulin obtained in A3:

[0098] Prepare 5% stacking gel and 12% separating gel:

[0099] Prepare 12% separating gel: Take 30% acrylamide / bisacrylamide solution, 1.5 mol / L Tris-HCl buffer (pH 8.8), distilled water, and 10% SDS in proportion, mix well. The total volume is calculated according to the required gel volume to prepare 12% separating gel solution. Add 10% ammonium persulfate to the separating gel solution, stir gently, then add TEMED, and mix quickly. Pour the mixed separating gel solution into the glass plate sandwich, seal with water, and let it stand until solidified.

[0100] Prepare 5% stacking gel: Take 30% acrylamide / bisacrylamide solution, 1.0 mol / L Tris-HCl buffer (pH 6.8), distilled water, and 10% SDS in proportion, mix well. The total volume is calculated according to the required gel volume to prepare 5% stacking gel solution. Add 10% ammonium persulfate to the stacking gel solution, stir gently, then add TEMED, and mix quickly. Suck off the water on the surface of the separating gel, pour the mixed stacking gel solution onto the solidified separating gel, insert the comb, and let it stand until solidified.

[0101] Accurately transfer 50 ng, 100 ng, 200 ng, 400 ng, and 800 ng of the denatured soy globulin obtained in step A3 into the gel comb holes. Make 5 replicates for each mass of soy globulin. Then add electrophoresis buffer to the inner and outer tanks of the electrophoresis tank respectively. Connect the power supply and perform constant voltage electrophoresis (70 V for stacking gel and 110 V for separating gel) until the bromophenol blue indicator migrates to 1 cm from the gel front, then stop electrophoresis to obtain the denatured soy globulin gel.

[0102] A5. Equilibrate the denatured soy glycinin gel obtained in A4 with transfer buffer for 30 min. Cut a PVDF membrane of appropriate size, soak it in absolute methanol for 10 s, rinse it with ultrapure water for 3 min, and transfer it to the transfer buffer to equilibrate for 30 min. Cut two pieces of Whatman filter paper slightly larger than the gel and soak them with the transfer buffer. Assemble each component on the transfer device, and the installation order from the negative electrode to the positive electrode is as follows: sponge pad, filter paper, denatured soy glycinin gel, PVDF membrane, filter paper, sponge pad. Add transfer buffer to the tank type electrotransfer device and transfer the membrane at 70 V for 2 h to obtain a denatured soy glycinin transfer membrane.

[0103] A6. Add the denatured soy glycinin transfer membrane obtained in A5 to the blocking buffer and incubate at room temperature for 2 h. Rinse it once with TBST buffer, add the rabbit anti-soy glycinin polyclonal antibody obtained in Example 3, and incubate overnight at 4 °C. Then wash the membrane three times with TBST buffer, 10 min each time. Then add horseradish peroxidase (HRP)-labeled anti-rabbit IgG antibody and incubate at room temperature for 2 h. Wash the membrane with TBST buffer, add chemiluminescent substrate for color development, record the picture, use an imaging system to collect the membrane image after color development, and use ImageJ analysis software to perform optical density analysis on each band to obtain the optical density value OD of each subunit or peptide chain of soy glycinin, calculate the loading amount of each subunit or peptide chain of soy glycinin, use the optical density value OD of each subunit or peptide chain as the independent variable, and use the loading amount (ng) of each subunit or peptide chain of soy glycinin as the dependent variable to establish a standard curve for detecting soy glycinin based on Western blotting technology.

[0104] Among them, the loading amount (W A ) of each subunit or peptide chain of soy glycinin is calculated as follows:

[0105]

[0106] Among them, W 1 is the loading amount of soy glycinin; A 1 is the optical density value of the soy glycinin subunit or peptide chain; A 0 is the total optical density value of the soy glycinin lane.

[0107] The specific test results are shown in Table 1 below:

[0108] Table 1 Loading amount and optical density value of each subunit or peptide chain of soy glycinin

[0109]

[0110]

[0111] The standard curve equations for detecting soy glycinin, A3 subunit, AS subunit, and BS subunit are respectively as Figures 1-4。

[0112] Example 5

[0113] Construct a standard curve for detecting β-conglycinin based on Western blotting technique. The construction method is the same as that in Example 4, with the differences being: in A1, the purified β-conglycinin prepared in Example 2 is selected, and in A2, the rabbit anti-β-conglycinin polyclonal antibody prepared in Example 3 is selected. Obtain the optical density value OD of each subunit or peptide chain of β-conglycinin, calculate the loading amount of each subunit or peptide chain of β-conglycinin. Taking the optical density value OD of each subunit or peptide chain as the independent variable and the loading amount (ng) of each subunit or peptide chain of β-conglycinin as the dependent variable, establish a standard curve for detecting β-conglycinin based on Western blotting technique.

[0114] The loading amount (W B ) of each subunit or peptide chain of β-conglycinin is calculated as follows:

[0115]

[0116] Among them, W 2 is the loading amount of β-conglycinin; A 1 is the optical density value of the β-conglycinin subunit or peptide chain; A 0 is the total optical density value of the β-conglycinin lane.

[0117] The specific test results are shown in Table 2:

[0118] Table 2 Loading amount and optical density value of each subunit or peptide chain of β-conglycinin

[0119]

[0120] The standard curves for detecting β-conglycinin, α'-subunit, α-subunit, and β-subunit are as Figures 5-8 。

[0121] Example 6

[0122] A construction method of a quantitative detection system for glycinin based on Western blotting technique includes the following steps:

[0123] S1. Use Example 4 to construct a standard curve for detecting glycinin based on Western blotting technique, calculate the content of glycinin, each subunit or peptide chain in the raw soybean sample, use Example 5 to construct a standard curve for detecting β-conglycinin based on Western blotting technique, and calculate the content of β-conglycinin, each subunit or peptide chain in the raw soybean sample as follows:

[0124] T1. Use a crusher to crush the raw soybeans to be tested, pass through a 60-mesh sieve, accurately weigh 1 g and put it into a small beaker. Add 5 mL of cold acetone at 4 °C, seal the beaker mouth with a sealing film, place it on a magnetic stirrer, stir at 500 rpm at room temperature for 1 h, let it stand for 30 min, discard the supernatant. Add 5 mL of cold acetone at 4 °C again, repeat the above process, change the liquid twice until the supernatant is clear, discard the supernatant, transfer the beaker to a fume hood, and let the remaining acetone air-dry naturally. Add 20 mL of extraction buffer to the above beaker, place it on a magnetic stirrer, stir and extract at 1000 rpm at room temperature for 1 h, centrifuge at 9000 g for 30 min at 4 °C, discard the precipitate, and obtain the total protein extract of raw soybeans;

[0125] T2. Add 1.0 mL of the total protein extract of raw soybeans obtained in step T1 to 0.25 mL of 5× loading buffer and mix, boil for 10 min to obtain the denatured-sample total protein extract;

[0126] T3. Perform SDS-polyacrylamide gel electrophoresis on the denatured-sample total protein extract obtained in step T2: Prepare a 5% stacking gel and a 12% separating gel in the same way as in step S4 of Example 2 above for preparing the separating gel and stacking gel. Pipette 10 μL of the denatured-sample total protein extract obtained in step T2 into the gel comb holes and make 3 replicates. Then add electrophoresis buffer to the inner and outer tanks of the electrophoresis tank respectively, turn on the power supply, and perform constant voltage electrophoresis (70 V for the stacking gel and 110 V for the separating gel) until the bromophenol blue indicator migrates to 1 cm from the gel front, and then stop electrophoresis.

[0127] T4. Equilibrate the denatured-sample total protein gel obtained in step T3 with transfer buffer for 30 min. Cut a piece of PVDF membrane of appropriate size, soak it in anhydrous methanol for 10 s, then rinse it with ultrapure water for 3 min, and transfer it to transfer buffer to equilibrate for 30 min. Cut two pieces of Whatman filter paper slightly larger than the gel, and soak them with transfer buffer. Assemble each component on the transfer device, and the installation order from the negative electrode to the positive electrode is as follows: sponge pad, filter paper, denatured-sample total protein gel, PVDF membrane, filter paper, sponge pad. Add transfer buffer to the tank-type electrotransfer device, transfer the membrane at 70 V for 2 h to obtain the denatured-sample total protein transfer membrane.

[0128] T5. Incubate the denatured-total protein transfer membrane obtained in step T4 in the blocking buffer at room temperature for 2 h, then rinse it once with TBST buffer, add the polyclonal antibody against glycinin or the polyclonal antibody against β-conglycinin obtained above, and incubate overnight at 4 °C. Then wash the membrane three times with TBST buffer, 10 min each time. Then add the horseradish peroxidase (HRP)-labeled anti-rabbit IgG antibody and incubate at room temperature for 2 h, wash the membrane with TBST buffer, add the chemiluminescent substrate for color development, record the picture, use the imaging system to collect the image of the developed membrane, and perform densitometric analysis on each band using the analysis software to obtain the optical density value OD of each subunit or peptide chain of raw soybeans. Select the standard curve for detecting glycinin based on Western blotting constructed in Example 4 to calculate the content of glycinin, each subunit or peptide chain in the raw soybean sample; select the standard curve for detecting β-conglycinin based on Western blotting constructed in Example 5 to calculate the content of β-conglycinin, each subunit or peptide chain in the raw soybean sample.

[0129] Calculate the content of soybean allergenic proteins and their subunits or peptide chains in the sample, and the calculation formula is:

[0130]

[0131] Where C is the content of soybean allergenic proteins and their subunits or peptide chains in the sample, with the unit of mg / g; m is the amount of soybean allergenic proteins and their subunits or peptide chains calculated using the standard curve equation, with the unit of ng; M is the mass of the sample weighed, with the unit of g; V is the total volume of the supernatant obtained by centrifuging the total protein of the sample extracted with the extraction buffer, with the unit of mL; v is the sample loading volume during electrophoresis, with the unit of μL.

[0132] S2. Repeatability verification test, including the following steps:

[0133] C1. Select raw soybean samples of the same batch and, according to the determination method of S1, repeat the determination of the content of glycinin and its subunits or peptide chains or the content of β-conglycinin and its subunits or peptide chains 10 times;

[0134] C2. Calculate the average value and standard deviation (SD) of the determination results;

[0135] C3. Calculate the relative standard deviation (RSD) based on the standard deviation to evaluate the repeatability and stability of the detection method.

[0136]

[0137] S3. Spike recovery verification test, including the following steps:

[0138] D1. Add 1 mg of the purified soybean allergenic protein standard to 1 g of raw soybean powder to form a spiked sample;

[0139] D2. According to the measurement method of S1, measure the content of soybean allergenic protein and its subunits or peptide chains in the spiked sample;

[0140] D3. Calculate the recovery rate of soybean allergenic protein and its subunits or peptide chains in the spiked sample.

[0141] Example Seven

[0142] The experimental scheme is the same as that of Example Six, with the difference being that 1 g of autoclaved soybean was weighed, and the content of soybean allergenic protein, each subunit or peptide chain in the autoclaved soybean sample was calculated.

[0143] Example Eight

[0144] The experimental scheme is the same as that of Example Six, with the difference being that 1 g of soybean meal was weighed, and the content of soybean allergenic protein, each subunit or peptide chain in the soybean meal was calculated.

[0145] Example Nine

[0146] The experimental scheme is the same as that of Example Six, with the difference being that 1 g of expanded soybean was weighed, and the content of soybean allergenic protein, each subunit or peptide chain in the expanded soybean was calculated.

[0147] For the above Examples Six - Nine, the results of the repeatability verification test and the spiked recovery rate verification test are shown in Table 3 below.

[0148] Table 3 Repeatability Results of the Quantitative Detection Method of Soybean Allergenic Protein Based on Western Blotting

[0149]

[0150]

[0151]

[0152] As can be seen from Table 3, the relative standard deviation of measuring different samples is relatively low, indicating that the measurement results of the quantitative detection method of soybean allergenic protein based on Western blotting have good reproducibility and high precision.

[0153] Table 4 Spiked Recovery Rates of the Quantitative Detection Method of Soybean Allergenic Protein Based on Western Blotting

[0154]

[0155]

[0156] As can be seen from Table 4, the spiked recoveries of the quantitative detection method for soybean allergenic proteins based on Western blotting were between 100.02 - 104.37, and the recoveries were close to complete, indicating that this detection method has high accuracy and reliability and can accurately determine the content of soybean allergenic proteins and their subunits or peptide chains in samples.

[0157] S4. Weigh 1 g of boiled soybeans, 1 g of microwave-treated soybeans, 1 g of defatted soybean meal, 1 g of fermented soybean meal, and 1 g of enzymatically hydrolyzed soybean meal respectively, and use the test method provided in step S1 to measure the content of soybean allergenic proteins and their subunits or peptide chains respectively. The measurement results are shown in Table 5.

[0158] Table 5 Measured values of soybean allergenic proteins and their subunits or peptide chains in different soybean products

[0159]

[0160]

[0161]

[0162] As can be seen from Table 5, there are significant differences in the contents of glycinin and β-conglycinin and their subunits or peptide chains in boiled soybeans, microwave-treated soybeans, defatted soybean meal, fermented soybean meal, and enzymatically hydrolyzed soybean meal. The contents of glycinin and β-conglycinin and their subunits or peptide chains in boiled soybeans, microwave-treated soybeans, and defatted soybean meal are relatively high, while those in fermented soybean meal and enzymatically hydrolyzed soybean meal are relatively low.

[0163] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for constructing a quantitative detection system for soybean allergenic protein based on Western blotting technology, characterized in that: The steps include: S1. Construct a standard curve for detecting soybean allergenic proteins based on Western blotting technology; S2, repeatability verification test; S3, spike recovery verification test; S4. Extract the total protein in the soybean product sample to be tested, perform quantitative analysis based on the standard curve, and calculate the content of soybean allergenic protein and its subunits or peptide chains in the sample.

2. The method according to claim 1, characterized in that: In S1, the method for constructing the standard curve for detecting soybean allergenic proteins based on Western blotting technology is as follows: A1. Separate and purify soybean allergenic protein; A2, using the soybean allergenic protein obtained in A1 as an immunogen to immunize rabbits, and obtaining polyclonal antibodies by affinity chromatography of the immune serum; A3, denaturing the purified soybean allergenic protein obtained in A1 to obtain denatured soybean allergenic protein; A4, subjecting the denatured soybean allergenic protein obtained in A3 to SDS-polyacrylamide gel electrophoresis to obtain a denatured soybean allergenic protein gel; A5, transferring the denatured-soy allergenic protein gel obtained in A4 to a PVDF membrane to obtain a denatured-soy allergenic protein transfer membrane; A6, blocking the denatured soybean allergenic protein transfer membrane obtained in A5 with blocking buffer, adding the polyclonal antibody obtained in A2, incubating overnight, washing the membrane, then adding horseradish peroxidase-labeled anti-rabbit IgG antibody for incubation, and adding chemiluminescent substrate for color development; The membrane images after color development were collected using a gel imaging system, and the optical density of each band was analyzed using analysis software to establish a standard curve for detecting soybean allergenic proteins based on protein blotting technology.

3. The method according to claim 1 or 2, characterized in that: In S1, the soybean allergenic protein is one or both of glycinin and β-conglycinin.

4. The method according to claim 2, characterized in that: In A1, the separation method for purifying soybean allergenic protein comprises the following steps: B1. Select soybeans with intact structure, crush them, and sieve them to obtain soybean powder; B2. Add 4°C cold acetone to the soybean powder obtained in B1, stir at 500 rpm for 1 hour at room temperature, let stand for 30 minutes, discard the supernatant, repeat the above process until the supernatant is clear, discard the supernatant, and naturally air-dry the precipitate to obtain defatted soybean powder; B3, crude extraction of soybean allergenic protein: adding extraction buffer to the defatted soybean powder obtained in B2, stirring and extracting, centrifuging and discarding the precipitate, adjusting the pH, standing and centrifuging, collecting precipitate A, adjusting the pH value of the supernatant, stirring, standing, collecting precipitate B, and the obtained precipitate A and precipitate B are crude extracted soybean allergenic protein; B4. Dissolve the crude soybean allergenic protein obtained in B3 in PBS buffer, heat it in a water bath to 25°C, add solid ammonium sulfate, fully dissolve it, collect the supernatant, heat it in a water bath to 25°C, continue to add solid ammonium sulfate, and collect the precipitate; B5. Add PBS buffer to the precipitate obtained in B4 to dissolve it, transfer it to a dialysis bag, dialyze it at 4°C for 48 hours, collect the dialysate, and lyophilize it to obtain purified soybean allergenic protein.

5. The method according to claim 1, characterized in that: In S2, the repeatability verification test comprises the following steps: C1. Select samples of soybean products from the same batch and repeatedly measure the content of soybean allergenic protein and its subunits or peptide chains; C2. Calculate the mean and standard deviation of the measurement results; C3. Calculate the relative standard deviation based on the standard deviation to evaluate the repeatability and stability of the detection method.

6. The method according to claim 1, characterized in that: In S3, the spike recovery verification test comprises the following steps: D1. Add a known amount of purified soy allergenic protein standard to the soy product sample to form a spiked sample; D2. Determine the content of soybean allergenic protein and its subunits or peptide chains in the spiked samples; D3. Calculate the recovery rate of soybean allergenic protein and its subunits or peptide chains in the spiked samples.

7. The method according to claim 1, characterized in that: In S4, the total protein in the soybean product sample to be tested is extracted, and quantitative analysis is performed according to the standard curve to calculate the content of soybean allergenic protein and its subunits or peptide chains in the sample. The calculation formula is: Wherein, C is the content of soybean allergenic protein and its subunits or peptide chains in the sample, in mg / g; m is the amount of soybean allergenic protein and its subunits or peptide chains calculated using the standard curve equation, in ng; M is the mass of the weighed sample, in g; V is the total volume of the supernatant obtained by adding extraction buffer to extract the total protein of the sample and centrifuging it, in mL; v is the amount of sample loaded during electrophoresis, in μL.

8. Use of the method according to any one of claims 1 to 7 in constructing a quantitative detection system for soybean allergenic protein based on Western blotting technology.

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