Method for analyzing sensitization effect of natural sugar chain on food allergen

Abstract

The invention relates to the technical field of food analysis, and provides a method for analyzing the sensitization effect of natural carbohydrate chains on food allergens, which comprises the following steps: extracting and purifying tropomyosin freeze-dried powder in food muscle tissues, identifying the types of carbohydrate chains in the tropomyosin freeze-dried powder, and analyzing the types of glucosidic bonds; removing an N-carbohydrate chain and an O-carbohydrate chain in the tropomyosin freeze-dried powder by using one or more of a PNGase F enzyme removal method, a beta-elimination method and a trifluoromethanesulfonic acid method, so as to obtain a deglycoprotein; a phenol-sulfuric acid method, SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) and PAS (Polyacrylamide Sulfate) dyeing are used for evaluating the anti An in vitro test and an in vivo test are used to evaluate the effect of the desugaring treatment on the allergenicity of tropomyosin. By analyzing the regulation influence of the natural carbohydrate chain on the allergenicity of the penaeus chinensis tropomyosin, a foundation is laid for deep research on the allergenicity of the penaeus chinensis tropomyosin, and an innovative perspective of analyzing the allergenicity characteristics of the penaeus chinensis tropomyosin is provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food analysis, and particularly relates to a method for analyzing the sensitization effect of natural sugar chains on food allergens. BACKGROUND

[0002] Food allergy is a global food safety and public health challenge, which continues to attract high attention from the scientific community and the public. Epidemiological investigations show that the incidence of food allergy is increasing. Shrimp is one of the eight major allergens and is a typical inducer of severe food allergy. Existing research shows that more than 80% of shrimp allergy cases are induced by tropomyosin (TM), a major allergen. As a typical member of the glycoprotein family, the natural glycosylation modification (including N-glycan and O-glycan structures) of TM is closely related to its allergenicity. At present, the research on tropomyosin in shrimp in the field mainly focuses on the reduction of epitopes and allergenicity, and there is little research on the influence of natural sugar chains on allergenicity. The regulatory effect of natural sugar chains on the allergenicity of shrimp tropomyosin has not been clearly defined, and therefore there is an urgent need in the field for a technical solution to explore the influence of natural sugar chains on the allergenicity of shrimp tropomyosin. SUMMARY

[0003] The purpose of the present application is to provide a method for analyzing the sensitization effect of natural sugar chains on food allergens. The method extracts and purifies tropomyosin (Pen c 1) from Chinese shrimp (Penaeus chinensis), analyzes the sugar chains of Pen c 1, and performs directed desugar chain processing. The desugar effect is evaluated by using key physicochemical property changes. Based on a three-dimensional evaluation system of serological detection (ELISA, Western blot), RBL-2H3 cell degranulation model and BALB / c mouse sensitization model, the regulatory effect of natural sugar chains on the allergenicity of Chinese shrimp tropomyosin is revealed.

[0004] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solution:

[0005] The present application provides a method for analyzing the sensitization effect of natural sugar chains on food allergens, which comprises the following steps:

[0006] (1) Extracting and purifying tropomyosin freeze-dried powder from muscle tissue of food;

[0007] (2) Identifying the types of sugar chains in the tropomyosin freeze-dried powder and analyzing the types of glycosidic bonds;

[0008] (3) Removing N-glycans and O-glycans in the tropomyosin freeze-dried powder using one or more of PNGase F desugarase, beta-elimination method and trifluoromethanesulfonic acid method to obtain desugar protein;

[0009] (4) evaluating the anti-sensitivity effect of the deglycosylated protein using phenol-sulfuric acid method, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and periodic acid-Schiff staining (PAS staining);

[0010] (5) evaluating the influence of the deglycosylation treatment on the sensitivity of tropomyosin using in vitro and in vivo tests.

[0011] Preferably, the extraction and purification method in step (1) is as follows:

[0012] A. mixing, stirring, and centrifuging the food and buffer 1, repeating the above operation 2-3 times to obtain a precipitate, defatting and drying the precipitate to obtain tissue powder;

[0013] B. mixing, extracting, and centrifuging the tissue powder and buffer 2 to obtain supernatant, purifying the supernatant using fractional ammonium sulfate precipitation method, heat denaturation purification method, and dialysis desalting method in sequence, quick-freezing at -85 to -75℃ for 20-30h, and freeze-drying to obtain a freeze-dried powder of tropomyosin.

[0014] Preferably, the buffer 1 in step A contains the following raw materials at the following final concentrations: 0.04-0.06mol / L potassium chloride and 0.01-0.03mol / L sodium bicarbonate, and the mixing ratio of the food and buffer 1 is 1g:9-11mL;

[0015] The mixing is performed 2-3 times, with an interval of 25-35s between each mixing, and the mixing speed is 9000-11000rpm;

[0016] The stirring is performed at a temperature of 2-6℃ for 3-5h;

[0017] The centrifugation is performed at a speed of 3000-5000r / min for 25-35min;

[0018] The defatting of the precipitate uses acetone, and the method is as follows: mixing the precipitate and acetone at a ratio of 1g:4-6mL, stirring at 2-6℃ for 5-7h, vacuum filtration, repeating until the filtrate is clear and the powder is milky white, drying the tissue powder in a fume hood overnight to obtain the tissue powder, the acetone is pre-cooled at -25 to -15℃, and the filter membrane used in the vacuum filtration has a specification of 0.45μm.

[0019] Preferably, the buffer 2 in step B contains the following raw materials at the following final concentrations: 0.8-1.2mol / L potassium chloride, 0.1-0.3mol / L tris-hydroxymethyl aminomethane, and 0.08-0.12mmol / L dithiothreitol;

[0020] The mixing ratio of the tissue powder and buffer 2 is 1g:19-21mL, and the pH of the mixture is 7-7.8;

[0021] The temperature of the extraction is 2-6℃;

[0022] The centrifugation speed is 8000-10000r / min, and the centrifugation time is 25-35min;

[0023] The specific steps of the fractional ammonium sulfate precipitation method are as follows: 15-25% saturated ammonium sulfate is used to precipitate impure proteins, the remaining solution is placed at 2-6℃ for 5-7h, and then centrifuged at 8000-10000r / min for 25-35min to remove the precipitate; the supernatant is used to precipitate the target protein with 35-45% saturated ammonium sulfate, and the solution is placed at 2-6℃ for 5-7h, then centrifuged at 8000-10000r / min for 25-35min to retain the precipitate; an appropriate amount of phosphate buffered saline (PBS buffer) is added to the precipitate and shaken until the precipitate is completely dissolved; the above operation is repeated 2-3 times.

[0024] The temperature of the heat denaturation purification method is 98-102℃, and the time is 8-12min; the temperature of the dialysis desalting method is 2-6℃, and the time is 45-50h.

[0025] Preferably, the method for identifying the type of sugar chain in the original myosin globulin freeze-dried powder in step (2) is glycosylation site prediction analysis; the method for analyzing the type of glycosidic bond is to specifically identify O-glycosidic bonds in the original myosin globulin freeze-dried powder by alkali treatment.

[0026] Preferably, the steps of the β-elimination method in step (3) are as follows: the original myosin globulin freeze-dried powder is dissolved in 0.8-1.2mg / mL potassium hydroxide solution, and reacted at 20-30℃ for 10-14h to obtain deglycosylated protein 1; the operation steps of the trifluoromethanesulfonic acid method are as follows: trifluoromethanesulfonic acid and anisole are mixed according to a volume ratio of 1-3:1 to prepare a deglycosylation reagent, the original myosin globulin freeze-dried powder is dissolved to 4-6mg / mL, and the reaction is carried out under nitrogen protection for 20-30min; a mixed solution prepared by mixing ether and n-hexane according to a volume ratio of 8-10:1 is added for precipitation, and the solution is placed at -25--15℃ for 3.5-4.5h, then centrifuged at 4000-6000×g for 10-20min; the precipitate is redissolved and dialyzed for 45-50h to obtain deglycosylated protein 2.

[0027] Preferably, the steps of the phenol-sulfuric acid method in step (4) are as follows:

[0028] A glucose standard curve was constructed, and the absorbance of the actomyosin freeze-dried powder and the deglycosylated protein at 480-500 nm was measured using a microplate reader. When the linear regression coefficient R2 of the glucose standard curve was greater than 0.99, the sugar content percentage of the actomyosin freeze-dried powder and the deglycosylated protein was calculated according to the following formula,

[0029]

[0030] As preferred, the method of the in vitro test in step (5) is one or several of Western Blot analysis, enzyme-linked immunosorbent assay analysis (ELISA analysis) and RBL-2H3 cell model establishment; and the method of the in vivo test is BALB / c mouse animal model establishment.

[0031] The application also provides application of the method in analyzing the sensitization effect of natural sugar chains on actomyosin.

[0032] The application has the following beneficial effects:

[0033] Since the regulatory effect of natural sugar chains on the sensitization of Chinese shrimp actomyosin (Pen c 1) is not clear, the present study takes the protein as the object, analyzes the sugar chain type and evaluates the deglycosylation effect. Bioinformatics analysis predicts that Pen c 1 contains 17 candidate N-glycosylation modification sites and 24 potential O-glycosylation modification sites, and PyMOL completes the spatial positioning annotation of these sites. Ultraviolet absorption experiment observes an absorption peak at 240 nm, confirming the presence of O-glycoside bonds in Pen c 1.

[0034] After deglycosylation treatment by the three methods, phenol-sulfuric acid method determination shows that Pen c 1 itself contains 2.550% of sugar, Pen c 1-DNG containing deglycosylated N-sugar chains contains 1.282% of sugar, Pen c 1-DOG containing deglycosylated O-sugar chains contains 1.125% of sugar, and Pen c 1-DNOG containing deglycosylated N&O-sugar chains contains 0.363% of sugar; SDS-PAGE electrophoresis shows that the molecular weight of Pen c 1 slightly decreases after deglycosylation, and PAS staining confirms that the deglycosylation effect is good, among which the triflic acid method is the most thorough.

[0035] In vitro and in vivo experiments verified the effect of natural sugar chain removal on Pen c 1 sensitization, and the results showed that desugarization would increase the sensitization. Western Blot / ELISA experiments showed that the binding capacity of desugared Pen c 1 to IgG and IgE antibodies was significantly improved; in the RBL-2H3 cell model, desugared Pen c 1 increased the cell degranulation efficiency, histamine content, and the contents of IL-4, IL-10, TNF-α, etc. In the BALB / c mouse experiment, desugared Pen c 1 caused more severe allergic symptoms in mice, more significant body temperature drop, and could significantly promote the secretion of Th2 type antibodies (IgE and IgG1) and the release of histamine and MCP-1 in serum; analysis of mouse spleen found that desugared Pen c 1 could enhance the spleen index and significantly increase the contents of IL-4, IL-10, and IL-13; H&E analysis showed that desugarization would make the inflammation symptoms in the lungs of mice more obvious. In addition, intestinal flora determination found that desugarization could differentially regulate the abundance of specific functional flora, increase the F / B ratio, and reduce Muribaculaceae and norank_f_Muribaculaceae. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 For structure visualization based on PyMOL and prediction of glycosylation site annotation;

[0037] Figure 2 For ultraviolet absorption spectrum analysis;

[0038] Figure 3 For sugar content analysis;

[0039] Figure 4 For SDS-PAGE molecular weight analysis, lane 1: Pen c 1; lane 2: Pen c 1-DNG; lane 3: Pen c 1-DOG; lane 4: Pen c 1-DNOG;

[0040] Figure 5 For PAS staining glycoprotein analysis, lane 1: Pen c 1; lane 2: Pen c 1-DNG; lane 3: Pen c 1-DOG; lane 4: Pen c 1-DNOG; lane 5: positive control-horseradish peroxidase; lane 6: negative control-soybean trypsin inhibitor;

[0041] Figure 6 For Western Blot analysis, lane 1: Pen c 1; lane 2: Pen c 1-DNG; lane 3: Pen c 1-DOG; lane 4: Pen c 1-DNOG;

[0042] Figure 7ELISA analysis for IgG binding capacity;

[0043] Figure 8 ELISA analysis for IgE binding capacity;

[0044] Figure 9 Analysis for degranulation efficiency;

[0045] Figure 10 Analysis for histamine release;

[0046] Figure 11 Analysis for IL-4 release;

[0047] Figure 12 Analysis for IL-13 release;

[0048] Figure 13 Analysis for TNF-α;

[0049] Figure 14 Mouse allergic sensitization and challenge experimental protocol;

[0050] Figure 15 Body temperature change curve;

[0051] Figure 16 Symptom score;

[0052] Figure 17 Serum IgE content analysis (A), serum IgG1 content analysis (B), serum IgG2a content analysis (C), serum histamine content analysis (D), serum MCP-1 content analysis (E);

[0053] Figure 18 Spleen index analysis (A), IL-4 content analysis (B), IL-10 content analysis (C), IL-13 content analysis (D), IFN-γ content analysis (E);

[0054] Figure 19 Mouse lung tissue section analysis, Note: 1: PBS; 2: Pen c 1; 3: Pen c 1-DNG; 4: Pen c 1-DOG; 5: Pen c 1-DNOG;

[0055] Figure 20 Mouse intestinal microbiota α-diversity index: Sobs index;

[0056] Figure 21 Mouse intestinal microbiota α-diversity index: Chao index;

[0057] Figure 22 Mouse intestinal microbiota α-diversity index: Shannon index;

[0058] Figure 23 The mouse intestinal microbiota alpha-diversity index: Simpson index;

[0059] Figure 24 The Venn diagram;

[0060] Figure 25 The taxonomic composition at the phylum level;

[0061] Figure 26 The taxonomic composition at the family level;

[0062] Figure 27 The taxonomic composition at the genus level. DETAILED DESCRIPTION

[0063] The present application provides a method for analyzing the sensitization effect of natural sugar chains on food allergens, comprising the following steps:

[0064] (1) extracting and purifying tropomyosin freeze-dried powder from food muscle tissue;

[0065] (2) identifying the types of sugar chains in the tropomyosin freeze-dried powder and analyzing the types of glycosidic bonds;

[0066] (3) removing N-glycan and O-glycan in the tropomyosin freeze-dried powder using one or more of PNGase F deglycosylation, beta-elimination method and trifluoromethanesulfonic acid method to obtain deglycosylated protein;

[0067] (4) evaluating the desensitization effect of the deglycosylated protein using phenol-sulfuric acid method, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and periodic acid-Schiff staining;

[0068] (5) evaluating the effect of deglycosylation on the allergenicity of tropomyosin using in vitro and in vivo tests.

[0069] In the present application, the method for extraction and purification in step (1) is preferably:

[0070] A. mixing, stirring and centrifuging the food and buffer 1, repeating the above operation 2-3 times to obtain a precipitate, defatting and drying the precipitate to obtain tissue powder;

[0071] B. mixing and leaching the tissue powder with buffer 2, centrifuging to obtain supernatant, purifying the supernatant sequentially using fractional ammonium sulfate precipitation method, heat denaturation purification method and dialysis desalting method, freezing at -85 to -75°C for 20-30h, and freeze-drying to obtain tropomyosin freeze-dried powder.

[0072] In the present application, the buffer 1 in step A preferably contains the following raw materials with the following final concentrations: 0.04-0.06 mol / L potassium chloride and 0.01-0.03 mol / L sodium bicarbonate, and further preferably 0.05 mol / L potassium chloride and 0.02 mol / L sodium bicarbonate; the mixing ratio of the food and the buffer 1 is preferably 1 g: 9-11 mL, and further preferably 1 g: 10 mL;

[0073] The mixing is performed 2-3 times, with an interval of 25-35 s between each mixing, and the mixing speed is 9000-11000 rpm; further preferably, the mixing is performed 3 times, with an interval of 30 s between each mixing, and the mixing speed is 10000 rpm;

[0074] The stirring temperature is 2-6℃, and the stirring time is 3-5 h; further preferably, the stirring temperature is 4℃, and the stirring time is 4 h;

[0075] The centrifugation speed is 3000-5000 r / min, and the centrifugation time is 25-35 min; further preferably, the centrifugation speed is 4000 r / min, and the centrifugation time is 30 min;

[0076] The solution used for the precipitation and defatting is preferably acetone; the method for the precipitation and defatting is preferably as follows: the precipitate and the acetone are mixed at a ratio of 1 g: 4-6 mL, and then stirred at 2-6℃ for 5-7 h, followed by vacuum filtration; the process is repeated until the filtrate is clear and the powder is milky white; the tissue powder is dried in a fume hood overnight to obtain the tissue powder; the acetone is pre-cooled at -25 to -15℃; and the filter membrane used for the vacuum filtration has a specification of 0.45 μm; further preferably, the method for the precipitation and defatting is as follows: the precipitate and the acetone are mixed at a ratio of 1 g: 5 mL, and then stirred at 4℃ for 6 h, followed by vacuum filtration; the process is repeated until the filtrate is clear and the powder is milky white; the tissue powder is dried in a fume hood overnight to obtain the tissue powder; the acetone is pre-cooled at -20℃; and the filter membrane used for the vacuum filtration has a specification of 0.45 μm.

[0077] In the present application, the buffer 2 in step B preferably contains the following raw materials with the following final concentrations: 0.8-1.2 mol / L potassium chloride, 0.1-0.3 mol / L tris-hydroxymethyl aminomethane, and 0.08-0.12 mmol / L dithiothreitol; and further preferably contains the following raw materials with the following final concentrations: 1.0 mol / L potassium chloride, 0.2 mol / L tris-hydroxymethyl aminomethane, and 0.1 mmol / L dithiothreitol;

[0078] The mixing ratio of the tissue powder and the buffer 2 is preferably 1 g: 19-21 mL, and further preferably 1 g: 20 mL; and the pH of the mixing is preferably 7-7.8, and further preferably 7.4;

[0079] The temperature of the leaching is preferably 2-6°C, and further preferably 4°C;

[0080] The centrifugal speed is 8000-10000r / min, and the centrifugal time is 25-35min; further preferably, the centrifugal speed is 9000r / min, and the centrifugal time is 30min;

[0081] The specific steps of the fractional ammonium sulfate precipitation method are preferably as follows: 15-25% saturated ammonium sulfate is used for removing impurity proteins, the remaining solution is placed at 2-6°C for 5-7h, and then centrifuged at 8000-10000r / min for 25-35min to remove the precipitate, and the supernatant is used for precipitating the target protein by using 35-45% saturated ammonium sulfate, and then placed at 2-6°C for 5-7h, and then centrifuged at 8000-10000r / min for 25-35min to retain the precipitate, and then the precipitate is dissolved by adding an appropriate amount of phosphate buffered saline, and the above operation is repeated 2-3 times; further preferably, 20% saturated ammonium sulfate is used for removing impurity proteins, the remaining solution is placed at 4°C for 6h, and then centrifuged at 9000r / min for 30min to remove the precipitate, and the supernatant is used for precipitating the target protein by using 40% saturated ammonium sulfate, and then placed at 4°C for 6h, and then centrifuged at 9000r / min for 30min to retain the precipitate, and then the precipitate is dissolved by adding an appropriate amount of phosphate buffered saline, and the above operation is repeated 2 times;

[0082] The temperature of the heat denaturation purification method is 98-102°C, and the time of the heat denaturation purification method is 8-12min; further preferably, the temperature of the heat denaturation purification method is 100°C, and the time of the heat denaturation purification method is 10min;

[0083] The temperature of the dialysis desalting method is 2-6°C, and the time of the dialysis desalting method is 45-50h; further preferably, the temperature of the dialysis desalting method is 4°C, and the time of the dialysis desalting method is 48h.

[0084] In the present application, the method for identifying the type of sugar chain in the original myosin globulin freeze-dried powder in step (2) is preferably glycosylation site prediction analysis; and the method for analyzing the type of glycosidic bond is preferably an alkali treatment method for specifically identifying O-glycosidic bonds in the original myosin globulin freeze-dried powder.

[0085] In the present application, the steps of the β-elimination method in step (3) are preferably as follows: the original myosin globulin freeze-dried powder is dissolved in a 0.8-1.2mg / mL potassium hydroxide solution, and reacted at 20-30°C for 10-14h to obtain deglycosylated protein 1; further preferably, the original myosin globulin freeze-dried powder is dissolved in a 1mg / mL potassium hydroxide solution, and reacted at 25°C for 12h to obtain deglycosylated protein 1;

[0086] The operation steps of the triflic acid method are preferably as follows: triflic acid and anisole are prepared into a desorption reagent with a volume ratio of 1-3:1, the original myosin lyophilized powder is dissolved to 4-6 mg / mL, a mixed solution prepared from ether and n-hexane with a volume ratio of 8-10:1 is added for precipitation under nitrogen protection for 20-30 min, and then centrifuged at 4000-6000xg for 10-20 min after standing at-25--15℃ for 3.5-4.5 h, and the precipitate is redissolved and dialyzed for 45-50 h to obtain the desorption protein 2; and further preferably, triflic acid and anisole are prepared into a desorption reagent with a volume ratio of 2:1, the original myosin lyophilized powder is dissolved to 5 mg / mL, a mixed solution prepared from ether and n-hexane with a volume ratio of 9:1 is added for precipitation under nitrogen protection for 25 min, and then centrifuged at 5000xg for 15 min after standing at-20℃ for 4 h, and the precipitate is redissolved and dialyzed for 48 h to obtain the desorption protein 2.

[0087] In the present application, the steps of the phenol-sulfuric acid method in step (4) are preferably as follows:

[0088] A glucose standard curve is constructed, and the absorbance of the original myosin lyophilized powder and the desorption protein at 480-500 nm is measured by using an enzyme marker; when the linear regression coefficient R2 of the glucose standard curve is greater than 0.99, the sugar content percentage of the original myosin lyophilized powder and the desorption protein is calculated according to the following formula, 2

[0089]

[0090] Further preferably, the steps of the phenol-sulfuric acid method in step (4) are as follows:

[0091] A glucose standard curve is constructed, and the absorbance of the original myosin lyophilized powder and the desorption protein at 490 nm is measured by using an enzyme marker; when the linear regression coefficient R2 of the glucose standard curve is greater than 0.99, the sugar content percentage of the original myosin lyophilized powder and the desorption protein is calculated according to the following formula,

[0092]

[0093] In the present application, the method of the in vitro test in step (5) is preferably one or more of Western blot analysis, enzyme-linked immunosorbent assay analysis and establishment of an RBL-2H3 cell model, and further preferably, Western blot analysis, enzyme-linked immunosorbent assay analysis and establishment of an RBL-2H3 cell model are used in sequence; and the method of the in vivo test is preferably establishment of a BALB / c mouse animal model.

[0094] The present application also provides application of the method in analysis of the effect of natural sugar chains on the allergenicity of myosin.

[0095] ​The technical solutions provided by the application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the application.

[0096] The application takes Chinese shrimp tropomyosin as an example to provide a method for analyzing the sensitization effect of natural sugar chains on food allergens, which comprises the following steps.

[0097] Example 1: Deglycosylation of Chinese shrimp tropomyosin and evaluation of its effect

[0098] 1) Extraction and purification of Chinese shrimp tropomyosin:

[0099] The head, shell and shelly line of fresh Chinese shrimp were removed to obtain washed muscle tissue. The muscle tissue (500 g) was mixed with Buffer A buffer 1 (0.05 mol / L potassium chloride, 0.02 mol / L sodium bicarbonate) at 1 g: 10 mL (w / v), homogenized 3 times (15 s / time, interval 30 s) by a homogenizer (10000 rpm), and then stirred at 4°C for 4 h. The precipitate was collected by centrifugation at 4000 r / min for 30 min, and Buffer A was repeated for three times. The final precipitate was defatted by acetone: 1 g: 5 mL (w / v) of pre-cooled acetone at-20°C was added, and then stirred at 4°C for 6 h. After vacuum filtration (0.45 μm filter), the process was repeated until the filtrate was clear and the powder was milky white. The obtained powder was dried in a fume hood overnight to obtain tissue powder.

[0100] The tissue powder was mixed with Buffer B buffer 2 (1 mol / L potassium chloride, 0.2 mol / L tris-hydroxymethyl aminomethane, 0.1 mmol / L dithiothreitol, pH 7.4) at 1 g: 20 mL (w / v) ratio, and then extracted overnight at 4°C. The next day, the supernatant was collected by centrifugation at 9000 r / min for 30 min. The fractionated ammonium sulfate precipitation method was used: first, 20% saturation degree was used to precipitate impurities, and then the precipitate was placed at 4°C for 6 h. After centrifugation at 9000 r / min for 30 min, the precipitate was removed, and the supernatant was used for 40% saturation degree to precipitate the target protein, and then the precipitate was placed at 4°C for 6 h. After centrifugation at 9000 r / min for 30 min, the precipitate was retained, and then an appropriate amount of phosphate buffered saline was added to the precipitate and shaken until the precipitate was completely dissolved. The precipitate-redissolution process was repeated for a total of 3 times. The supernatant was further purified by heat stability treatment (100°C, 10 min), and then dialyzed at 4°C for 48 h by using a 3 kDa dialysis bag. After dialysis, the sample was quickly frozen at-80°C for one day, and then freeze-dried to obtain the final tropomyosin.

[0101] Example 2: Analysis of sugar chains in Chinese shrimp tropomyosin freeze-dried powder

[0102] 1) Analysis of the type of sugar chains of Pen c 1

[0103] The amino acid sequence of Pen c 1 (GenBank: ADA70137.1) was subjected to glycosylation site prediction analysis by NetNGlyc 1.0 Server (website: http: / / www.cbs.dtu.dk / services / NetNGlyc / ) and NetOGlyc 4.0 Server (website: http: / / www.cbs.dtu.dk / services / NetOGlyc / ), respectively. Among them, NetNGlyc 1.0 is used for N-glycosylation site identification, and NetOGlyc 4.0 is used for O-glycosylation site detection. Structure visualization and prediction of glycosylation site annotation were performed using PyMOL, see Figure 1 .

[0104] 2) Glycosidic bond analysis

[0105] To analyze the linkage mode of the sugar chain of Pen c 1, the alkali treatment method was used to specifically identify the O-glycosidic bond: Pen c 1 protein solution (0.2 mg / mL, 90 μL) was mixed with 0.5 mol / L NaOH (10 μL), and incubated at 20°C for 16 h. At the same time, the Pen c 1 solution without alkali treatment was used as a control, and the ultraviolet-visible spectrophotometer was used to scan the 200-280 nm absorption spectrum. The O-glycosidic bond in the alkali-treated Chinese shrimp tropomyosin freeze-dried powder was verified by comparing the absorbance change at 245 nm.

[0106] Conclusion: Since the regulatory effect of natural sugar chains on the allergenicity of Chinese shrimp tropomyosin has not been clear, the present application takes Chinese shrimp tropomyosin as the research object, analyzes the type of sugar chain, and evaluates the desugarization effect by desugarization treatment. The results show that based on bioinformatics analysis and prediction, 17 candidate N-glycosylation modification sites and 24 potential O-glycosylation modification sites are located in the Pen c 1 protein sequence. Further, spatial positioning annotation of the predicted glycosylation sites was completed using PyMOL. With the aid of ultraviolet absorption experiment (see Figure 2 ), an absorption peak was observed at 240 nm, which confirmed the presence of O-glycosidic bond in Pen c 1.

[0107] Example 3

[0108] 1) Pen c 1 desugarization treatment

[0109] ① Take Pen c 1 protein freeze-dried powder in a centrifuge tube, and use PNGase F desugarase to remove the N-glycan of Pen c 1 to obtain Pen c 1-DNG;

[0110] Pen c 1 was dissolved in 1 mg / mL potassium hydroxide solution by β-elimination method, and reacted at 25°C for 12 h, and Pen c 1-DOG was obtained by dialysis.

[0111] ②Pen c 1-DOG was obtained by removing the O-glycan of Pen c 1 by β-elimination method;

[0112] Pen c 1 was dissolved in 1 mg / mL potassium hydroxide solution by β-elimination method, and reacted at 25°C for 12 h, and Pen c 1-DOG was obtained by dialysis.

[0113] ③Pen c 1-DNOG was obtained by removing the N-glycan and O-glycan of Pen c 1 by trifluoromethanesulfonic acid method;

[0114] The operation of trifluoromethanesulfonic acid method was carried out in a fume hood. The desugar reagent was prepared according to trifluoromethanesulfonic acid: anisole = 2:1 (v / v), Pen c 1 was dissolved (5 mg / mL), and the reaction was carried out under nitrogen protection for 25 min. Ethyl ether:n-hexane (9:1 v / v) was added to precipitate the protein, and the precipitate was centrifuged (5000 x g, 15 min) after standing at -20°C for 4 h. The precipitate was redissolved and dialyzed for 48 h to obtain Pen c 1-DNOG.

[0115] 2) Evaluation of Pen c 1 desugar effect

[0116] ① Phenol-sulfuric acid method: accurately construct a glucose standard curve: take 0.1 mol / L glucose standard according to 0 (blank), 4, 8, 16, 32, 64, 128, 200 μL into 5 mL centrifuge tube, and add ultrapure water to 200 μL per tube, and set three parallel groups for each concentration. Take 200 μL of dialyzed and purified Pen c 1 solution before and after desugar chain for parallel treatment, and add 120 μL of pre-cooled 6% phenol solution (w / v) and 600 μL of concentrated sulfuric acid to all samples in turn, vortex for 30 s, mix well, and heat in a boiling water bath for 20 min, and immediately terminate the reaction in an ice bath. Use the enzyme marker to measure the absorbance at 490 nm. Only when the linear regression coefficient R2 of the standard curve is greater than 0.99, calculate the sugar content percentage of Pen c 1 and its desugar treated protein according to the formula, see Figure 3 .

[0117]

[0118] ② SDS-PAGE: Take 20 μL of Pen c 1 solution after dialysis purification and 5x protein loading buffer in a 4:1 volume ratio, heat in a 100℃ water bath for 7 min. With a vertical electrophoresis system, install 12% Tris-Glycine precast gel, add electrophoresis buffer to the inner tank liquid surface to cover the gel upper edge. Accurately load 7 μL Marker and 2 μg protein sample per well. Initially set 80V constant voltage to make the sample pass through the concentrated gel, and when the bromophenol blue migrates to the interface of the separation gel, adjust to 120V constant voltage for continuous electrophoresis. When the tracking dye is 5 mm from the bottom of the gel, terminate the electrophoresis, and use microwave-assisted Coomassie brilliant blue staining method (60℃ heating for 1 min, 50 r / min shaking for 3 min) for protein color development, then decolorization, finally collect the gel image by Image Lab 6.0 system, see Figure 4 .

[0119] ③ PAS staining: To clarify the natural sugar chain modification characteristics of Pen c 1, PAS staining method was used to detect all samples in parallel. According to the sample processing method of SDS-PAGE, 0.5 μg / μL horseradish peroxidase (HRP) was used as a positive control sample for glycoprotein detection, and soybean trypsin inhibitor (STI) was used as a negative control sample. Strictly follow the operation specification of the kit instruction, sequentially carry out gel fixation (40% ethanol / 5% acetic acid), periodic acid oxidation (1% solution, 4℃ dark for 30 min), Schiff reagent staining (room temperature shaking for 45 min) and metabisulfite elution. Finally, the staining results were obtained by gel imaging system, see Figure 5 .

[0120] Conclusion: The three deglycosylation methods were used to verify the deglycosylation effect. The phenol-sulfuric acid method was used to determine the sugar content, and it was found that the sugar content of Penc 1 itself was 2.550%, the sugar content of Pen c 1-DNG after removing N-glycan was 1.282%, the sugar content of Pen c 1-DOG after removing O-glycan was 1.125%, and the sugar content of Pen c 1-DNOG after removing N&O-glycan was 0.363%. The SDS-PAGE electrophoresis results showed that the molecular weight of Pen c 1 after deglycosylation decreased, but the change was not significant. PAS staining further confirmed that the effect of deglycosylation was good, especially the trifluoromethanesulfonic acid method for removing glycan was the most thorough.

[0121] Example 4 Effect of deglycosylation on in vitro allergenicity of tropomyosin

[0122] 1) Western Blot analysis

[0123] Respectively, 20 μL of Pen c 1 protein sample and its deglycosylation modification products (Pen c 1-DNG, Pen c1-DOG, Pen c 1-DNOG) were mixed with 5 times concentrated SDS-PAGE loading buffer according to the volume ratio of 4:1 as the experimental group. The mixed sample system was placed in a 100℃ water bath environment for denaturation treatment for 7 min. The electrophoresis device was used for electrophoresis separation under the condition of constant voltage 120V, and terminated when the bromophenol blue indicator migrated to the bottom of the gel. The PVDF membrane (0.45 μm) was cut to 8.6x6.4 cm, activated with anhydrous methanol for 30 s, and then rinsed with ultrapure water for 3 times (1 min each time). The semi-dry transfer system was used for transfer under the condition of constant voltage 25V for 10 min. The four channels of the full-automatic protein blotting system were placed in the cleaning tank, and 7 empty centrifuge tubes were placed in the reagent bin. The system clean program was run for cleaning. Respectively, 10 mL of primary antibody working solution (rabbit anti-TM antibody diluted with blocking solution at 1:10000) and secondary antibody working solution (HRP labeled goat anti-rabbit IgG diluted with blocking solution at 1:10000) were prepared, and the primary antibody, secondary antibody and blocking solution were placed in order according to the system label. The membrane was placed in the system according to the sandwich method, the edit button was clicked, the parameters were set, the start button of the channel was clicked, and the instrument started to run. After 7 h of system reaction, the membrane was taken out and transferred to the white plate of the gel imaging instrument for 1 min staining. Then the gel imaging system was used to image and take pictures of the sample strip and Marker respectively for saving, as shown in Figure 6 .

[0124] 2) ELISA analysis

[0125] After dilution of Pen c 1 and its deglycosylation products (Pen c 1-DNG, Pen c 1-DOG, Pen c 1-DNOG) to a final concentration of 5 μg / mL using CBS (carbonate-bicarbonate buffer), 100 μL / well was added to a 96-well plate for coating, and incubated in a constant temperature incubator at 37°C for 2 h. Three washes (300 μL / well / time, each for 30 s) were performed using PBST (phosphate buffered saline containing Tween-20), followed by the addition of 200 μL / well of blocking solution, and continued blocking for 2 h under the same conditions. After performing the above-mentioned PBST washing procedure for 3 times again, 100 μL / well of the following diluted antibodies was added: rabbit anti-TM IgG (1:5,000 dilution) and mouse anti-TM IgE working solution. Incubation was performed at 37°C for 1 h. After washing, the corresponding HRP-labeled secondary antibody (1:10,000) was added, and after 1 h of reaction under constant temperature conditions at 37°C, three repeated washes were performed using PBST. After the reaction was completed, 100 μL / well of TMB color developing substrate solution was added, and color developing reaction was performed in the dark for 15 min. After the color developing reaction was terminated, 50 μL / well of stop solution was immediately added, and finally the absorbance value at 450 nm was detected by an enzyme-labeled instrument. The IgG antibody binding capacity and IgE antibody binding capacity of the deglycosylation products were respectively Figure 7 and Figure 8 .

[0126] 3) RBL-2H3 cell model

[0127] Establishment of RBL-2H3 mast cell model: the cells were cultured in MEM medium (minimum essential medium) containing 10% FBS, and cultured in a cell incubator (37°C, 5% CO2), and subcultured every 2-3 days; the cell suspension concentration was adjusted to 1×10 6After 24h, the cells were washed twice with PBS and incubated with 100 μL of fresh medium containing 1 :80 diluted mouse serum for 12h. At the end of the incubation, the medium was replaced with basal medium and the cells were incubated with 100 μL of 10 μg / mL of Pen c 1, Pen c 1 -DNG, Pen c 1 -DOG or Pen c 1 -DNOG for 1 h. The supernatants were collected and 100 μL of 0.5% Triton-X 100 lysis solution was added to each well for 15 min at room temperature in the dark. The supernatants and lysis solutions were transferred to centrifuge tubes and centrifuged at 1,000 rpm for 5 min at 4°C to remove cell debris. The supernatants were used for the determination of histamine and cytokine levels. The lysis solutions were used for the determination of the degranulation rate. Figure 9 Degranulation rate = (OD405 of the sample - OD405 of the control) / (OD405 of the maximum - OD405 of the control) x 100

[0128]

[0129] In addition, the amount of histamine (see Figure 10 ) and the amount of cytokines (IL-4, IL-13) (see Figure 11 , Figure 12) using commercial ELISA kit, and the detailed operation steps are described in the kit instruction. The cytokine TNF-a was not detected in the supernatant of RBL-2H3 after Pen c 1 stimulation, so the lysis solution of RBL-2H3 was used to measure the protein concentration according to the commercial ELISA kit instruction, and the standard sample was added to the 96-well plate at 0 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, 20 μL per well, and the protein concentration in the lysis solution was measured. The relative expression amount was calculated according to the value of the Pen c 1 group as the root, and the relative expression amount was calculated according to the value of the Pen c 1 group as the root. Figure 13 The relative expression amount calculation formula is:

[0130]

[0131] Example 5 Effect of deglycosylation on in vivo sensitization of tropomyosin

[0132] BALB / c mouse animal model was established (see Figure 14 ): 6-week-old female BALB / c mice were raised in a SPF level animal room. The experimental animal room maintained a constant temperature and humidity environment (temperature 25±2℃, relative humidity 50±5%), an automatic controlled light system was used to ensure the 12h / 12h light-dark rhythm, and non-allergenic mouse feed was used for feeding, and the drinking water was changed regularly every day. After 7 days of adaptive feeding, the formal experiment was started. In the experimental setup, 50 female BALB / c mice were randomly assigned to 5 groups (n=10), and the specific grouping was as follows: PBS group (as negative control), Pen c 1 group, Pen c 1-DNG group, Pen c 1-DOG group and Pen c 1-DNOG group. During the sensitization stage (i.e. on days 0, 7, 14, 21, 28 of the experiment), the experimental groups received intraperitoneal injection of 200 μL suspension containing 100 μg antigen (dissolved in 100 μL aluminum hydroxide adjuvant), and the control group was injected with the same volume of sterile PBS (pH 7.4). 72 hours after the last immunization, serum samples were obtained by tail vein blood collection, and then the ELISA was used to quantitatively detect the titer level of IgE type antibody in the serum. When the detected IgE titer reached or exceeded the preset threshold value of 1:6400, the experiment would proceed to the challenge stage. When the experiment was carried out to the 35th day, the experimental group mice received 1 mg of antigen (dissolved in 200 μL PBS) by oral gavage, while the control group received the same volume of PBS solution. After gavage, all mice were fasted overnight. Note that after all operations, the mice were measured for body temperature Figure 15 ) and symptom score Figure 16). The next morning, the enucleation of eyeball was performed for blood collection. The collected blood samples were centrifuged at 3000 r / min for 10 min at 4℃, and after centrifugation, the serum was aliquoted and stored at -80℃ for subsequent monitoring of specific antibodies (IgE, IgG1, IgG2a), histamine and MCP-1 Figure 17 ) in serum. Meanwhile, the mice were dissected, and the tissue samples were quickly removed, and the spleen index was calculated Figure 18 ), and the spleen was aliquoted in 1640 medium for monitoring of cytokines IL-4, IL-10, IL-13, IFN-γ in lymphocytes, and the lung tissue was perfused and fixed with 4% paraformaldehyde for H&E staining (see Figure 19 ), and the feces were taken for determination of intestinal flora (see Figures 20-27 .

[0133] Conclusion: Examples 4 and 5 verified the influence of removal of natural sugar chains on sensitization by constructing in vitro and in vivo tests. It was found that the removal of sugar chains could increase the sensitization in both in vivo and in vitro tests. It was found that the binding capacity of Pen c 1 treated by deglycosylation to IgG and IgE antibodies was significantly improved by Western Blot / ELISA experiment. The RBL-2H3 cell model showed that the degranulation efficiency, histamine content and cytokine (IL-4, IL-10, TNF-α) content were increased after Pen c 1 deglycosylation. The BALB / c mouse experiment found that the allergic symptoms were more severe after Pen c 1 deglycosylation, and the body temperature drop was more significant. It was found that Pen c 1 treated by deglycosylation could significantly promote the secretion of Th2 type antibodies (IgE and IgG1) and the release of histamine and MCP-1 in serum. The analysis of mouse spleen showed that Pen c 1 treated by deglycosylation could enhance the spleen index and significantly increase the content of cytokines (IL-4, IL-10, IL-13). The H&E analysis results showed that the lung inflammation symptoms became more obvious with the treatment of deglycosylation. In addition, the determination of intestinal flora in mouse feces showed that the deglycosylation treatment differentially regulated the abundance of specific functional flora, increased the F / B ratio, and was accompanied by the decrease of Muribaculaceae and norank_f_Muribaculaceae.

[0134] From the above embodiments, the application provides a method for analyzing the sensitization effect of natural sugar chains on food allergens, comprising the following steps: extracting and purifying tropomyosin freeze-dried powder in food muscle tissue, identifying the types of sugar chains therein, and analyzing the types of glycosidic bonds; removing N-glycan and O-glycan in the tropomyosin freeze-dried powder by using one or more of PNGase F deglycosylation enzyme, beta-elimination method and trifluoromethanesulfonic acid method to obtain deglycosylated protein; using phenol-sulfuric acid method, SDS-PAGE and PAS staining to evaluate the desensitization effect of the deglycosylated protein; using in vitro and in vivo tests to evaluate the influence of deglycosylation treatment on the sensitization of tropomyosin. The application analyzes the regulatory influence of natural sugar chains on the sensitization of Chinese shrimp tropomyosin, constructs a method for analyzing the sensitization effect of natural sugar chains on glycoprotein allergens, lays a foundation for in-depth research on the sensitization of Chinese shrimp tropomyosin, and provides an innovative perspective for analyzing the sensitization characteristics of Chinese shrimp tropomyosin. At the same time, the application provides a new strategy for studying allergen sensitization and provides new evidence for the regulatory role of glycosylation modification in food allergy.

[0135] The above only describes the preferred embodiments of the application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the application, and these improvements and refinements should also be considered within the protection scope of the application.

Claims

1. A method for analyzing the sensitizing effect of natural sugar chains on food allergens, characterized in that, The method includes the following steps: (1) Extraction and purification of tropomyosin freeze-dried powder from food muscle tissue; (2) Identify the types of sugar chains in tropomyosin freeze-dried powder and analyze the types of glycosidic bonds; (3) Use one or more of PNGase F desaccharidase, β-elimination method and trifluoromethanesulfonic acid method to remove N-glycans and O-glycans from tropomyosin lyophilized powder to obtain desaccharified protein; (4) The anti-sensitivity effect of deglycoprotein was evaluated by phenol-sulfuric acid method, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and periodic acid-Schiff staining. (5) The effects of desaccharification treatment on tropomyosin sensitization were evaluated using in vitro and in vivo tests.

2. The method according to claim 1, characterized in that, The extraction and purification method described in step (1) is as follows: A. Mix the food and buffer solution 1, stir, and centrifuge. Repeat the above operation 2-3 times to obtain a precipitate. Defatt the precipitate and dry it to obtain tissue powder. B. Mix the tissue powder and buffer 2, extract, and centrifuge to obtain the supernatant. Purify the supernatant sequentially using fractional ammonium sulfate precipitation, thermal denaturation purification, and dialysis desalting. Quick-freeze at -85 to -75°C for 20 to 30 hours and freeze-dry to obtain tropomyosin lyophilized powder.

3. The method according to claim 2, characterized in that, The buffer solution 1 in step A contains the following raw materials at final concentrations: 0.04-0.06 mol / L potassium chloride and 0.01-0.03 mol / L sodium bicarbonate, and the ratio of the food to buffer solution 1 is 1 g: 9-11 mL; The mixing is performed 2 to 3 times, with an interval of 25 to 35 seconds between each mixing, and the mixing speed is 9000 to 11000 rpm. The stirring temperature is 2-6℃, and the stirring time is 3-5 hours. The centrifugation speed is 3000-5000 r / min, and the centrifugation time is 25-35 min; The precipitate degreasing solution used is acetone. The precipitation degreasing method is as follows: the precipitate and acetone are mixed at a ratio of 1g:4-6mL, stirred at 2-6℃ for 5-7h, and then vacuum filtered. This process is repeated until the filtrate is clear and the powder is milky white. The tissue powder is then dried overnight in a fume hood to obtain the tissue powder. The acetone used is pre-cooled acetone at -25 to -15℃, and the filter membrane used for vacuum filtration has a specification of 0.45μm.

4. The method according to claim 2, characterized in that, The buffer solution 2 described in step B contains the following raw materials at final concentrations: 0.8–1.2 mol / L potassium chloride, 0.1–0.3 mol / L tris(hydroxymethyl)aminomethane, and 0.08–0.12 mmol / L dithiothreitol; The ratio of the tissue powder to buffer 2 is 1g:19-21mL, and the pH of the mixture is 7-7.

8. The extraction temperature is 2–6°C; The centrifuge speed is 8000-10000 r / min, and the centrifugation time is 25-35 min; The specific steps of the graded ammonium sulfate precipitation method are as follows: 15-25% saturated ammonium sulfate precipitation is used to remove impurities and proteins. The remaining solution is allowed to stand at 2-6℃ for 5-7 hours, then centrifuged at 8000-10000 r / min for 25-35 minutes to remove the precipitate. The supernatant is retained and the target protein is precipitated with 35-45% saturated ammonium sulfate. The solution is allowed to stand at 2-6℃ for 5-7 hours, then centrifuged at 8000-10000 r / min for 25-35 minutes. The precipitate is retained, and an appropriate amount of phosphate buffered saline is added to the precipitate and shaken until the precipitate is completely dissolved. The above operation is repeated 2-3 times. The temperature of the heat denaturation purification method is 98–102°C, and the time of the heat denaturation purification method is 8–12 min; The temperature for the dialysis desalination method is 2–6°C, and the dialysis desalination time is 45–50 h.

5. The method according to claim 1, characterized in that, The method for identifying the glycan type in tropomyosin lyophilized powder in step (2) is glycosylation site prediction analysis; the method for analyzing the types of glycosidic bonds is to specifically identify O-glycosidic bonds in tropomyosin lyophilized powder using an alkaline treatment method.

6. The method according to claim 1, characterized in that, The steps of the β-elimination method described in step (3) are as follows: dissolve the lyophilized tropomyosin powder in a potassium hydroxide solution of 0.8-1.2 mg / mL, react at 20-30℃ for 10-14 h, and dialyze to obtain deglycosylated protein 1; The operation steps of the trifluoromethanesulfonic acid method are as follows: Trifluoromethanesulfonic acid and anisole are mixed in a volume ratio of 1 to 3:1 to prepare a desaccharification reagent, the lyophilized tropomyosin powder is dissolved to 4 to 6 mg / mL, and the reaction is carried out under nitrogen protection for 20 to 30 min. A mixed solution of diethyl ether and n-hexane in a volume ratio of 8 to 10:1 is added for precipitation. After standing at -25 to -15℃ for 3.5 to 4.5 h, the mixture is centrifuged at 4000 to 6000 × g for 10 to 20 min. After redissolving the precipitate, it is dialyzed for 45 to 50 h to obtain desaccharified protein 2.

7. The method according to claim 1, characterized in that, The steps of the phenol-sulfuric acid method described in step (4) are as follows: A glucose standard curve was constructed, and the absorbance of tropomyosin lyophilized powder and deglycosylated protein at 480–500 nm was measured using an ELISA reader. When the linear regression coefficient R² of the glucose standard curve was greater than 0.99, the percentage of sugar content in tropomyosin lyophilized powder and deglycosylated protein was calculated using the following formula.

8. The method according to claim 1, characterized in that, The in vitro test method mentioned in step (5) is one or more of the following: Western blot analysis, enzyme-linked immunosorbent assay (ELISA) analysis, and the establishment of an RBL-2H3 cell model; the in vivo test method is the establishment of a BALB / c mouse animal model.

9. The application of the method according to any one of claims 1 to 8 in analyzing the sensitizing effect of natural glycans on tropomyosin.

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