Ulva agglutinin as well as extraction method and application thereof

Abstract

The invention discloses ulva agglutinin as well as an extraction method and application thereof. The extraction method comprises the following steps: S1, grinding dried ulva into ulva powder; s2, uniformly mixing the ulva powder with a NaCl-containing phosphate buffer solution, extracting ulva agglutinin by an ammonium sulfate precipitation method after ultrasonic-assisted extraction, so as to prepare an ulva agglutinin crude extract; and S3, purifying the ulva agglutinin crude extract by adopting an AKTA avat25 protein purification system, so as to obtain the ulva agglutinin. The ulva agglutinin has high sensitization, an RBL-2H3 cell model is established, and the sensitization of the ulva agglutinin is evaluated through the influence of the ulva agglutinin on the cell morphology or the cell histamine release rate. By combining ammonium sulfate precipitation with ion exchange chromatography, the purity of the ulva agglutinin is improved and is obviously superior to that in the prior art, and the method is more efficient and higher in purity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of preparation and safety evaluation of marine bioactive proteins, and in particular to a genus chondrus agglutinin and an extraction method and application thereof. BACKGROUND

[0002] Chondrus is a large green algae widely distributed in the intertidal zone of global temperate to tropical coasts. In recent years, it has been regarded as a potential functional food raw material and sustainable biological resource due to its rapid growth, large biomass, and rich content of proteins, polysaccharides, minerals, and various bioactive substances. In Asian countries, Chondrus has been used as a traditional food material for hundreds of years, and its application value in the fields of bioenergy, medicine, and cosmetics has also attracted widespread attention. The umami amino acids contained in Chondrus add a unique umami flavor to seaweed, and Chondrus can be processed into food with unique flavor, realizing the food utilization of Chondrus. Chondrus also contains active substances that can be used as additives, such as polyphenols extracted from Chondrus, which have antioxidant, antibacterial, and fresh-keeping effects on food such as meat and can be used as harmless natural food additives. Chondrus agglutinin has attracted attention in the fields of functional food development (such as dietary supplements) and disease treatment (such as antitumor adjuvants) due to its unique sugar binding activity and immunomodulatory function. However, the large-scale preparation and safety evaluation of Chondrus agglutinin still face the following technical bottlenecks: 1) the traditional extraction process is low in efficiency and insufficient in purity, and existing Chondrus agglutinin extraction methods (such as dialysis and low-temperature ethanol precipitation) have problems such as low protein recovery rate and high impurity residue, which leads to insufficient reliability of subsequent functional research; 2) the allergenic risk of marine agglutinin proteins is unclear, and the systematic allergenic risk evaluation method for Chondrus agglutinin, a type of marine agglutinin protein, is still in the blank state, and the potential allergenicity of Chondrus agglutinin and its mechanism have not been clarified. SUMMARY

[0003] In view of the above problems existing in the prior art, the present application provides a genus chondrus agglutinin and an extraction method and application thereof. The present application improves the purity of Chondrus agglutinin by ammonium sulfate precipitation combined with ion exchange chromatography, which is significantly superior to the prior art, more efficient and higher in purity, and a rapid allergenicity evaluation method based on in vitro cell models for marine agglutinin is constructed.

[0004] The technical scheme of the present application is as follows:

[0005] The first object of the present application is to provide an extraction method of Chondrus agglutinin, comprising the following steps:

[0006] S1: grinding dry Chondrus into Chondrus powder;

[0007] S2: mixing the Ulva lactuca powder with the phosphate buffer containing NaCl, extracting the Ulva lactuca lectin from the mixture by ultrasonic-assisted extraction and ammonium sulfate precipitation to obtain the crude extract of the Ulva lactuca lectin;

[0008] S3: purifying the crude extract of the Ulva lactuca lectin by using an AKTA avant25 protein purification system to obtain the Ulva lactuca lectin.

[0009] In an embodiment of the present application, in step S2, the concentration of NaCl in the phosphate buffer containing NaCl is 0.15 mol / L, the concentration of Na2HPO4-NaH2PO4 is 0.015 mol / L, and the pH is 7.2.

[0010] In an embodiment of the present application, in step S2, the ratio of the Ulva lactuca powder to the phosphate buffer containing NaCl is 1:15 g / mL.

[0011] In an embodiment of the present application, in step S2, the power of the ultrasonic-assisted extraction is 120-160 W.

[0012] In an embodiment of the present application, in step S2, the process of extracting the Ulva lactuca lectin by ammonium sulfate precipitation is as follows: first, 20% saturation (NH4)2SO4 treatment, then 60-75% saturation (NH4)2SO4 treatment, and finally dialysis to obtain the crude extract of the Ulva lactuca lectin.

[0013] In an embodiment of the present application, in step S3, the mobile phase of the AKTA avant25 protein purification system is 50 mM Tris-HCl with a pH of 7.0-8.0; the concentration of NaCl in the elution phase is 0-1 M, and the flow rate of the elution phase is 1 mL / min.

[0014] In an embodiment of the present application, in step S3, the concentration of the intermediate product crude extract loaded on the column is 5-10 mg / mL.

[0015] The measured yield of the Ulva lactuca lectin is 1.33±0.16%.

[0016] The second object of the present application is to provide the Ulva lactuca lectin prepared by the above extraction method.

[0017] In an embodiment of the present application, the Ulva lactuca lectin has high sensitization.

[0018] In an embodiment of the present application, the evaluation method of the sensitization of the Ulva lactuca lectin comprises the following steps:

[0019] (1) establishing an RBL-2H3 cell model;

[0020] (2) qualitative evaluation of the sensitization of the lectin by observing the morphological changes of the cells treated with the lectin;

[0021] (3) quantitative evaluation of the sensitization of the lectin by observing the histamine release rate of the cells treated with the lectin.

[0022] In one embodiment of the present application, in steps (1) and (2), the RBL-2H3 cells are inoculated in a 12-well cell culture plate at a density of 5.0×10 4 / well for 24 h, and the cells are treated with the sample to be tested for 24 h, and then stained with toluidine blue for 30 min, and the cell morphology is observed under an inverted microscope and photographed.

[0023] In one embodiment of the present application, in steps (1) and (3), the cells are inoculated in a 96-well cell culture plate at a density of 1×10 4 / well for 24 h, and the cells are treated with the sample to be tested for 24 h, and then the supernatant of the cell culture is removed and centrifuged at 1000×g for 20 min, and the supernatant is taken and used to detect the histamine level in the supernatant of the cells by using a Histamine ELISA kit.

[0024] A third object of the present application is to provide the above-mentioned lectin for use as an internal standard for allergen detection.

[0025] The present application has the following beneficial technical effects:

[0026] The present application improves the extraction method of the lectin, which is more efficient and has higher purity, and a rapid sensitization evaluation method based on an in vitro cell model is constructed. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The figure is the yield of the lectin in Example 1, Comparative Example 1 and Comparative Example 2.

[0028] Figure 2 The figure is the yield of the lectin in Example 2, Comparative Example 3 and Comparative Example 4.

[0029] Figure 3 The figure is the purity of the lectin in Example 2 and Comparative Example 5.

[0030] Figure 4 The figure is the SDS-PAGE of the lectin in Example 1 and Comparative Examples 1-2.

[0031] Figure 5 The figure is the SDS-PAGE of the lectin in Example 2 and Comparative Examples 3-4.

[0032] Figure 6SDS-PAGE figure of the Ulva pertusa agglutinin in Comparative Example 5.

[0033] Figure 7 SDS-PAGE figure of the Ulva pertusa agglutinin in Example 2.

[0034] Figure 8 Cell morphology figure after treating RBL-2H3 cells with the intermediate product in Example 2, the final product in Example 2 and the Ulva pertusa agglutinin in Comparative Example 5.

[0035] Figure 9 Cell histamine content figure after treating RBL-2H3 cells with the intermediate product in Example 2, the final product in Example 2 and the Ulva pertusa agglutinin in Comparative Example 5. DETAILED DESCRIPTION

[0036] The present application will be described in detail below with reference to the accompanying drawings and examples.

[0037] The Ulva pertusa in the examples of the present application is from Yantai, Shandong Province, and is purchased from the Qingdao Changdao Seafood Store.

[0038] Example 1

[0039] A method for extracting an Ulva pertusa agglutinin, comprising the following steps:

[0040] S1: Dry Ulva pertusa is ground into Ulva pertusa powder in a grinder, and is stored in a dry and dark place for later use;

[0041] S2: 50 g of the Ulva pertusa powder is added into a phosphate buffer solution containing NaCl (0.15 mol / L NaCl, 0.015 mol / L Na2HPO4-NaH2PO4, pH 7.2) according to a material-to-liquid ratio of 1:15 (g / mL), and is subjected to ultrasonic treatment for 10 min in an ultrasonic disruptor with an ultrasonic power of 160 W;

[0042] S3: The sample after S2 is placed in a 4℃ chromatography cabinet, is stirred with a magnetic stirrer for 16 h, and is centrifuged at a speed of 9000 rpm for 20 min in a high-speed refrigerated centrifuge to obtain a crude extract of the Ulva pertusa agglutinin;

[0043] S4: The crude extract of the Ulva pertusa agglutinin in S3 is placed in a 4℃ chromatography cabinet, and solid (NH4)2SO4 is added to make the crude extract of the Ulva pertusa agglutinin reach 20% saturation. After stirring with a magnetic stirrer for 12 h, the mixture is centrifuged at a speed of 11000 rpm for 20 min in a high-speed refrigerated centrifuge to discard the precipitate and take the supernatant. Solid (NH4)2SO4 is added to the supernatant to make it reach 75% saturation in a 4℃ chromatography cabinet, and after stirring with a magnetic stirrer for 12 h, the mixture is centrifuged at a speed of 11000 rpm for 20 min in a high-speed refrigerated centrifuge. The precipitate is collected and is re-dissolved in distilled water.

[0044] S5: After dialysis of the sample in S4 in deionized water for 48h with a 3500Da dialysis bag, freeze-drying is performed in a freeze-drier;

[0045] S6: A Tris-HCl mobile phase (50mM Tris, pH 7.7) and a Tris-HCl-NaCl elution phase (50mM Tris, 1M NaCl, pH 7.7) are prepared;

[0046] S7: The sample obtained in S5 is dissolved in the mobile phase to obtain a sample solution of 8mg / mL, and the sample solution is filtered with a 0.45μm filter membrane to obtain a sample for loading;

[0047] S8: The sample in S7 is separated by an AKTA avant 25 protein purification system, eluted with the elution phase at a flow rate of 1mL / min, through 3 column volumes, and the components are collected, desalted by centrifugation at 5050rpm for 15min in a high-speed refrigerated centrifuge with a 1000Da ultrafiltration tube, and then freeze-dried in a freeze-drier to obtain the purified Ulva agglutinin.

[0048] Example 2:

[0049] A method for extracting Ulva agglutinin, comprising the following steps:

[0050] S1: Dry Ulva is ground into Ulva powder in a grinder, and stored in a dry and dark place for use;

[0051] S2: 50g of Ulva powder is added to a phosphate buffer containing NaCl (0.15mol / L NaCl, 0.015mol / L Na2HPO4-NaH2PO4, pH 7.2) at a solid-liquid ratio of 1:15 (g / mL), and ultrasonic treatment is performed in an ultrasonic crusher at an ultrasonic power of 120W for 10min;

[0052] S3: The sample after S2 treatment is placed in a 4℃ chromatography cabinet and stirred with a magnetic stirrer for 16h, and then centrifuged at a speed of 9000rpm for 20min in a high-speed refrigerated centrifuge to obtain a crude Ulva agglutinin extract;

[0053] S4: The crude Ulva pertussin solution in S3 was placed in a 4°C chromatography cabinet, and solid (NH4)2SO4 was added to make the crude Ulva pertussin solution reach 20% saturation. After being stirred with a magnetic stirrer for 12 h, the solution was centrifuged at 11000 rpm for 20 min in a high-speed refrigerated centrifuge. The precipitate was discarded, and the supernatant was added with solid (NH4)2SO4 to reach 60% saturation at 4°C in the chromatography cabinet. After being stirred with a magnetic stirrer for 12 h, the solution was centrifuged at 11000 rpm for 20 min in a high-speed refrigerated centrifuge. The precipitate was collected and redissolved in distilled water;

[0054] S5: The redissolved sample in S4 was dialyzed in a 3500 Da dialysis bag in deionized water for 48 h, and then freeze-dried in a freeze dryer;

[0055] S6: A Tris-HCl mobile phase (50 mM Tris, pH 7.7) and a Tris-HCl-NaCl elution phase (50 mM Tris, 1 M NaCl, pH 7.7) were prepared;

[0056] S7: The sample obtained in S5 was dissolved in the mobile phase to obtain a sample solution of 8 mg / mL. The sample solution was filtered through a 0.45 μm filter membrane to obtain a sample for loading;

[0057] S8: The sample in S7 was separated by an AKTA avant 25 protein purification system at a flow rate of 1 mL / min, eluted with the elution phase, and collected after 3 column volumes. The components were desalted by centrifugation at 5050 rpm for 15 min in a high-speed refrigerated centrifuge using a 1000 Da ultrafiltration tube, and then freeze-dried in a freeze dryer to obtain purified Ulva pertussin.

[0058] Example 3:

[0059] The same as Example 1, except that in step S2, the ultrasonic power was 140 W; and in step S6, the pH of the mobile phase was 8.0.

[0060] Comparative Example 1:

[0061] S1: A certain amount of dried Ulva was ground into Ulva powder in a grinder, and stored in a dry and dark place for use. 50 g of the Ulva powder was taken.

[0062] S2: The Ulva powder was added to a phosphate buffer containing NaCl (0.15 mol / L NaCl, 0.015 mol / L Na2HPO4-NaH2PO4, pH 7.2) at a solid-liquid ratio of 1:10 (g / mL) in an ultrasonic crusher, and ultrasonic treatment was performed at an ultrasonic power of 160 W for 10 min.

[0063] S3: The sample after S2 was placed in a 4℃ chromatography cabinet and stirred with a magnetic stirrer for 16h, and then centrifuged at 9000rpm for 20min in a high-speed refrigerated centrifuge to obtain a crude extract of Ulva pertussa agglutinin.

[0064] S4: The crude extract of Ulva pertussa agglutinin in S3 was placed in a 4℃ chromatography cabinet, and solid (NH4)2SO4 was added to make the crude extract of Ulva pertussa agglutinin reach 20% saturation. After stirring with a magnetic stirrer for 12h at 4℃, the mixture was centrifuged at 11000rpm for 20min in a high-speed refrigerated centrifuge, and the precipitate was discarded and the supernatant was collected. Solid (NH4)2SO4 was added to the supernatant to reach 75% saturation at 4℃, and then the mixture was stirred with a magnetic stirrer for 12h at 4℃, and then centrifuged at 11000rpm for 20min in a high-speed refrigerated centrifuge. The precipitate was collected and redissolved in distilled water.

[0065] S5: The redissolved sample in S4 was dialyzed in a 3500Da dialysis bag in deionized water for 48h, and then freeze-dried in a freeze dryer.

[0066] Comparative Example 2:

[0067] S1: A certain amount of dried Ulva pertussa was ground into Ulva pertussa powder in a grinder, and stored in a dry and dark place for use. 50g of Ulva pertussa powder was taken.

[0068] S2: The Ulva pertussa powder was added to a phosphate buffer containing NaCl (0.15mol / L NaCl, 0.015mol / L Na2HPO4-NaH2PO4, pH 7.2) at a solid-to-liquid ratio of 1:20 (g / mL), and treated with ultrasonic waves in an ultrasonic crusher at an ultrasonic power of 160W for 10min.

[0069] S3: The sample after S2 was placed in a 4℃ chromatography cabinet and stirred with a magnetic stirrer for 16h, and then centrifuged at 9000rpm for 20min in a high-speed refrigerated centrifuge to obtain a crude extract of Ulva pertussa agglutinin.

[0070] S4: The crude Ulva pertussin solution in S3 was placed in a 4°C chromatography cabinet, and solid (NH4)2SO4 was added to make the crude Ulva pertussin solution reach 20% saturation. After stirring with a magnetic stirrer for 12 h, the solution was centrifuged in a high-speed refrigerated centrifuge at 11000 rpm for 20 min. The precipitate was discarded, and the supernatant was taken. Solid (NH4)2SO4 was added to the supernatant to make it reach 75% saturation, and it was placed in a 4°C chromatography cabinet. After stirring with a magnetic stirrer for 12 h, the solution was centrifuged in a high-speed refrigerated centrifuge at 11000 rpm for 20 min. The precipitate was collected and redissolved in distilled water.

[0071] S5: The redissolved sample in S4 was dialyzed in a 3500 Da dialysis bag in deionized water for 48 h, and then freeze-dried in a freeze dryer.

[0072] Comparative Example 3:

[0073] S1: A certain amount of dried Ulva was ground into Ulva powder in a pulverizer and stored in a dry and dark place for use. 50 g of the Ulva powder was taken.

[0074] S2: The Ulva powder was added to a phosphate buffer containing NaCl (0.15 mol / L NaCl, 0.015 mol / L Na2HPO4-NaH2PO4, pH 7.2) at a solid-to-liquid ratio of 1:15 (g / mL), and ultrasonic treatment was performed in an ultrasonic crusher with an ultrasonic power of 80 W for 10 min.

[0075] S3: The sample treated in S2 was placed in a 4°C chromatography cabinet and stirred with a magnetic stirrer for 16 h. Then, the solution was centrifuged in a high-speed refrigerated centrifuge at 9000 rpm for 20 min to obtain a crude Ulva pertussin solution.

[0076] S4: The crude Ulva pertussin solution in S3 was placed in a 4°C chromatography cabinet, and solid (NH4)2SO4 was added to make the crude Ulva pertussin solution reach 20% saturation. After stirring with a magnetic stirrer for 12 h, the solution was centrifuged in a high-speed refrigerated centrifuge at 11000 rpm for 20 min. The precipitate was discarded, and the supernatant was taken. Solid (NH4)2SO4 was added to the supernatant to make it reach 60% saturation, and it was placed in a 4°C chromatography cabinet. After stirring with a magnetic stirrer for 12 h, the solution was centrifuged in a high-speed refrigerated centrifuge at 11000 rpm for 20 min. The precipitate was collected and redissolved in distilled water.

[0077] S5: The redissolved sample in S4 was dialyzed in a 3500 Da dialysis bag in deionized water for 48 h, and then freeze-dried in a freeze dryer.

[0078] Comparative Example 4:

[0079] S1: A certain amount of dried Enteromorpha prolifera was ground into powder in a grinder and stored in a dry and dark place for later use. 50 g of the powder was taken.

[0080] S2: The powder was added to a phosphate buffer containing NaCl (0.15 mol / L NaCl, 0.015 mol / L Na2HPO4-NaH2PO4, pH 7.2) at a solid-to-liquid ratio of 1:15 (g / mL), and was subjected to ultrasonic treatment for 10 min in an ultrasonic disrupter set at an ultrasonic power of 160 W.

[0081] S3: The sample treated in S2 was placed in a 4°C chromatography cabinet and stirred with a magnetic stirrer for 16 h, and then was centrifuged at a speed of 9000 rpm for 20 min in a high-speed refrigerated centrifuge to obtain a crude extract of Enteromorpha prolifera lectin.

[0082] S4: The crude extract of Enteromorpha prolifera lectin in S3 was placed in a 4°C chromatography cabinet, and solid (NH4)2SO4 was added to make the crude extract reach a saturation degree of 20%. After stirring with a magnetic stirrer for 12 h at 4°C, the mixture was centrifuged at a speed of 11000 rpm for 20 min in a high-speed refrigerated centrifuge, and the precipitate was discarded and the supernatant was collected. Solid (NH4)2SO4 was added to the supernatant to make it reach a saturation degree of 60% at 4°C, and then the mixture was stirred with a magnetic stirrer for 12 h at 4°C, and was centrifuged at a speed of 11000 rpm for 20 min in a high-speed refrigerated centrifuge. The precipitate was collected and was re-dissolved in distilled water.

[0083] S5: The re-dissolved sample in S4 was dialyzed in a 3500 Da dialysis bag in deionized water for 48 h, and was then freeze-dried in a freeze dryer.

[0084] Comparative Example 5

[0085] S1-S7 were the same as in Example 2.

[0086] S8: The sample in S7 was separated by an AKTA avant 25 protein purification system, and was eluted with a mobile phase at a flow rate of 1 mL / min. After 3 column volumes, the components were collected, desalted by centrifugation at a centrifugal force of 3000 x g for 15 min in a high-speed refrigerated centrifuge using a 1000 Da ultrafiltration tube, and then was freeze-dried in a freeze dryer to obtain Comparative Example 5.

[0087] Detection Test

[0088] 1. Take the intermediate product in S5 of Example 1, Example 2 and the final product of Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and use the BCA protein concentration determination kit to determine the lectin yield: prepare the BCA working solution by adding 1 volume of Cu reagent (50:1) to 50 volumes, dilute the sample, add 20 μL to the sample well of the 96-well enzyme-labeled plate, and then add 200 μL of the BCA working solution to each well, and place at 37°C for 30 min. Measure the absorbance at 562 nm using an enzyme-labeled instrument, and then calculate the protein concentration according to the standard curve. Each sample is tested in triplicate to avoid experimental error. The results are shown in Table 1. Figure 1 , Figure 2 The lectin yield of the intermediate product in S5 of Example 1 is significantly higher than that of Comparative Example 1 and Comparative Example 2. The lectin yield of the intermediate product in S5 of Example 2 is significantly higher than that of Comparative Example 3 and Comparative Example 4.

[0089] 2. Take the intermediate product in S5 of Example 2, the final product of Example 2 and the final product of Comparative Example 5, and use the plant lectin kit to determine the lectin purity: add 50 μL of the sample to be tested to the sample well, add 50 μL of biotin-labeled antibody to each well, seal the reaction well with a sealing film, incubate in a 37°C incubator for 30 min, then discard the liquid and dry on absorbent paper, add enough washing solution to each well, stand for 1 min, then shake off the washing solution and dry on absorbent paper, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each sample well, seal the reaction well with a sealing film, and incubate in a 37°C incubator for 30 min. Then discard the liquid and dry on absorbent paper, add enough washing solution to each well, stand for 1 min, then shake off the washing solution and dry on absorbent paper, and repeat the washing step 5 times. Add 50 μL of substrate A and B to each well, and incubate at 37°C in the dark for 15 min. Then add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm within 15 min. Calculate the lectin purity of the intermediate product in S5 of Example 2, the final product of Example 2 and the final product of Comparative Example 5 according to the standard curve. The results are shown in Table 2. Figure 3 The lectin purity of the intermediate product in S5 of Example 2, the final product of Example 2 and the final product of Comparative Example 5 is significantly higher than that of Comparative Example 5.

[0090] 3. The intermediate product in S5 of Example 1, the intermediate product in S5 of Example 2, the final product of Example 2, and the samples of Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 were taken for determination of protein composition by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): using a sample dissolving solution containing 0.25M Tris-HCl, 20% glycerol and 4% SDS (pH 8.0), the protein concentration of each concentration sample was adjusted to 2 mg / mL. At the same time, 2% (w / v) (g / mL) bromophenol blue was added to the solution as an electrophoresis indicator to monitor the electrophoresis process. In the gel electrophoresis experiment stage, the electrophoresis system was constructed by preparing 12.5% separation gel and 3% concentrated gel. 8 μL Marker and 10 μL treated protein sample were added to the sample well, respectively, and the electrophoresis current was set to 12 mA. When the sample migrated to the bottom of the gel plate under the action of the electric field, the electrophoresis was terminated. After electrophoresis, the gel was stained with Coomassie brilliant blue R-250, and then rinsed with deionized water and decolorizing solution in turn. After the background color was fully removed, the gel was placed in a chemiluminescence imager for image acquisition and analysis, so as to obtain the protein composition information of the sample. Then the Quantity One software was used for protein molecular weight analysis. The results are shown in Figure 4 , Figure 5 , Figure 6 , Figure 7 Figure 4 For the SDS-PAGE results of the intermediate product in S5 of Example 1 and Comparative Example 1, Comparative Example 2, it can be seen that the distribution of lectin protein is less different. Figure 5 For the SDS-PAGE results of the intermediate product in S5 of Example 2 and Comparative Example 3, Comparative Example 4, compared with Comparative Example 3 and Comparative Example 4, the electrophoresis band of the intermediate product in S5 of Example 2 is clearer, and the protein with a molecular weight of about 18 kDa is the most. Figure 7 For the SDS-PAGE results of the intermediate product in S5 of Example 2 and the final product of S5 of Example 2, the electrophoresis band distribution of the intermediate product in S5 of Example 2 is 107 kDa, 75 kDa, 35 kDa, 23 kDa, 18 kDa and 11 kDa. The electrophoresis band distribution of the final product of S5 of Example 2 is 107.55, 75, 67.6, 35, 32.02, 28.75, 23.51, 18.7 and 10.7 kDa. The known lectin molecular weight is 35.54, 35.66, 105.01, 106.08 and 251.14 kDa. It is shown that the intermediate product and the final product of Example 2 both contain lectin.

[0091] ​4. Take the intermediate product from S5 of Example 2, the final product from Example 2, and the final product from Comparative Example 5, and treat RBL-2H3 cells grown to the logarithmic growth phase respectively, and observe the effect on the morphology of RBL-2H3 cells: RBL-2H3 cells were treated with 5.0 × 10⁻⁶ cells... 4 Cells were seeded at a density of / wells in 12-well cell culture plates and cultured for 24 h. Cells were then treated with 300 μg / mL of A, B, and Ulva lectins, respectively, for 24 h. Afterward, cells were stained with toluidine blue for 30 min, and cell morphology was observed and photographed under an inverted microscope. Results are as follows: Figure 8 As shown, the cells treated with the final product of Comparative Example 5 exhibited a long spindle shape, which is the normal morphology of cells. However, the cells treated with the intermediate product in S5 of Example 2 and the final product of Example 2 showed certain changes in morphology, changing from a spindle-shaped and slender shape to a round shape. The normal morphology of RBL-2H3 cells is long spindle-shaped, and the change in cell roundness indicates that the intermediate product in S5 of Example 2 and the final product of Example 2 stimulated the cells, causing their morphology to change.

[0092] The intermediate product from S5 of Example 2, the final product of Example 2, and the final product of Comparative Example 5 were used to treat RBL-2H3 cells grown to the logarithmic growth phase, respectively. The amount of histamine released from the RBL-2H3 cells was then detected. 4 Cells were seeded at a density of / wells in 96-well cell culture plates and cultured for 24 h. Then, cells were treated with 300 μg / mL of A, B, and Ulva lectins, respectively, for 24 h. The cell culture supernatant was then aspirated, centrifuged at 1000×g for 20 min, and the supernatant was used to detect histamine levels using a Histamine ELISA kit. Results are as follows: Figure 9 As shown, compared with the histamine release after treatment with the final product of Comparative Example 5, the histamine release rate of RBL-2H3 cells induced by the S5 intermediate product of Example 2 and the final product of Example 2 was significantly increased, reaching 42.40 ± 1.47 ng / mL, respectively. -1 and 45.28±2.40 ng / mL -1 The results showed that the intermediate product in S5 of Example 2 and the final product of Example 2 had stronger sensitizing properties.

[0093] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for extracting a stone-wort agglutinin, characterized by, The method comprises the following steps: S1: grinding dry Enteromorpha into Enteromorpha powder; S2: mixing the Enteromorpha powder with NaCl-containing phosphate buffer, and extracting Enteromorpha agglutinin by ultrasonic-assisted extraction and ammonium sulfate precipitation to obtain crude Enteromorpha agglutinin extract; S3: purifying the crude Enteromorpha agglutinin extract by AKTA avant 25 protein purification system to obtain the Enteromorpha agglutinin.

2. The extraction method according to claim 1, characterized in that, In step S2, the concentration of NaCl in the NaCl-containing phosphate buffer is 0.15 mol / L, the concentration of Na2HPO4-NaH2PO4 is 0.015 mol / L, and the pH is 7.

2.

3. The extraction method of claim 1, wherein, In step S2, the solid-liquid ratio of the Enteromorpha powder to the NaCl-containing phosphate buffer is 1:

15.

4. The extraction method of claim 1, wherein, In step S2, the ultrasonic-assisted extraction power is 120-160 W.

5. The extraction method of claim 1, wherein, In step S2, the process of extracting Enteromorpha agglutinin by ammonium sulfate precipitation is as follows: first 20% saturation (NH4)2SO4 treatment, then 60-75% saturation (NH4)2SO4 treatment, and finally dialysis, to obtain the crude Enteromorpha agglutinin extract.

6. The extraction method of claim 1, wherein, In step S3, the mobile phase of the AKTA avant 25 protein purification system is 50 mM Tris-HCl with pH 7.0-8.0; the concentration of NaCl in the elution phase is 0-1 M, and the flow rate of the elution phase is 1 mL / min.

7. Enteromorpha agglutinin prepared by the extraction method of any one of claims 1-6.

8. The Ulva pertussis agglutinin according to claim 7, characterized in that, Highly allergenic.

9. The Ulva pertussis agglutinin according to claim 7, characterized in that, The evaluation method of allergenicity comprises the following steps: (1) establishing an RBL-2H3 cell model; (2) qualitatively evaluating the allergenicity of Enteromorpha agglutinin by its influence on cell morphology; (3) quantitatively evaluating the allergenicity of Enteromorpha agglutinin by its influence on cell histamine release rate.

10. The use of the Ulva pertusa agglutinin according to claim 7, characterized in that, Internal standard for allergen detection.

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