A method for extracting and purifying animal digestive tract mucin

Abstract

A method for extracting and purifying mucin from the digestive tract of an animal, the present invention relates to a method for extracting and purifying mucin. The present invention solves the problem that the existing methods for extracting and purifying mucin from the digestive tract of an animal cannot achieve the best effect. Method: solution preparation, obtaining mucus samples, extraction and preparation of crude mucus extracts, and mucin extraction and purification. The present invention prolongs the incubation time of cysteine ​​protease inhibitors, and then ensures the freshness and activity of the extracted protein as much as possible under the condition of reducing the concentration gradient of cysteine ​​protease inhibitors, achieving an effect consistent with the protein extraction concentration. The present invention reduces the speed of centrifugation, thereby reducing the difficulty of extraction in terms of instrument selection and operability. The method of the present invention ensures the freshness of the protein, and the electrophoresis detection results show that there is no tailing phenomenon, the extraction effect is good, and the extraction purity is high.

Description

Technical Field

[0001] The invention relates to a method for extracting and purifying mucin. Background Art

[0002] The mucus layer covers the epithelial cells of the digestive tract. The integrity of the mucus barrier it constitutes is the first line of defense to protect the gastrointestinal tract. It can protect the gastrointestinal tract from pathogens and chemical and physical damage, and is crucial to the health and function of the gastrointestinal tract. Mucin is the main macromolecular glycoprotein in mucus. Its main function is to maintain the integrity and functionality of the intestinal mucosal barrier by binding to adhesion molecules on the surface of intestinal mucosal epithelial cells. Mucin not only provides a physical protective barrier for the homeostasis of the intestinal environment, but also participates in the intestinal immune system. Studies have found that by regulating the expression and composition of mucin, the balance of the intestinal microbiome, the secretion of antimicrobial peptides, and the activity of immune cells can be regulated, which helps to maintain intestinal health. In addition, some studies have also found that changes in the structure and function of mucin may be associated with intestinal inflammation and the development of other intestinal diseases. Therefore, in-depth research on mucin activity and regulatory mechanisms plays an important role in studying the digestive tract health, nutritional regulation technology, and intestinal environment of humans and animals, and can be applied to research in nutrition, animal development, and medicine. However, in current research, due to the difficulty of mucin purification methods, ultra-high-speed low-temperature centrifuges are required for long centrifugation times, and the extraction efficiency is low and difficult. As a result, research on mucins has only remained at the stage of detecting the expression levels of related mucins, and has been unable to conduct in-depth research, such as mucus co-culture and other subsequent verification experiments on mucus structure and function, and has been unable to achieve the best results. Summary of the invention

[0003] In order to solve the problem that the existing methods for extracting and purifying animal digestive tract mucin cannot achieve the best effect, the present invention provides a method for extracting and purifying animal digestive tract mucin.

[0004] The method for extracting and purifying animal digestive tract mucin of the present invention is carried out according to the following steps:

[0005] 1. Obtaining mucus samples: When sampling, rinse the intestinal segment with phosphate buffered saline (PBS), gently scrape the intestinal segment to obtain mucus and phosphate buffered saline (PBS) at a weight ratio of 1:5, mix them by centrifugation to retain the precipitate, and then add 5 times the volume of phosphate buffered saline (PBS) to the precipitate to obtain the mucus sample;

[0006] 2. Add 5 times the volume of the cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB mixed solution to the mucus sample obtained in step 1 and mix evenly, the volume ratio of cysteine ​​protease inhibitor CPI to membrane extraction buffer MEB is 2:1, and incubate overnight at 2-6°C to obtain sample A;

[0007] 3. Sample A was placed in a centrifuge tube, centrifuged for 10-80 min at a temperature of 0-6°C and a relative centrifugal force of 15000-30000g, and the supernatant was discarded; 5 times the volume of a mixture of cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB was added and mixed evenly, the volume ratio of cysteine ​​protease inhibitor CPI to membrane extraction buffer MEB was 2:1, and centrifuged at a temperature of 0-6°C and a relative centrifugal force of 15000-30000g, and the supernatant was discarded to obtain precipitate B;

[0008] 4. The precipitate B obtained in step 3 is placed in a centrifuge tube and centrifuged for 10-80 minutes at a temperature of 0-6°C and a relative centrifugal force of 15000-30000g, and the supernatant is discarded; 5 volumes of a mixture of cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB are added and mixed evenly, the volume ratio of cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB is 2:1, and the supernatant is discarded and the precipitate is retained by centrifugation at a temperature of 0-6°C and a relative centrifugal force of 15000-30000g. The precipitate repeats the process of centrifugation described in this step, adding CPI and MEB mixture and centrifuging again 1-2 times to obtain precipitate C;

[0009] 5. Fill the centrifuge tube containing the precipitate C with membrane extraction buffer MEB, add 50 μL of 2 mol / L dithiothreitol DTT to each tube per 1 mL of membrane extraction buffer MEB, fill to a final concentration of 100 mmol / L dithiothreitol DTT, and incubate overnight on a water bath shaker at 30-40°C; add 50 μL of 2 mol / L dithiothreitol DTT to each 1 mL of membrane extraction buffer MEB, and incubate on a water bath shaker at 30-40°C for 5-15 hours to obtain sample solution D;

[0010] 6. Add 300 μL 1.5 mol / L iodoacetamide IAA to every 1.5 mL sample solution D, store in dark, and incubate overnight in a 20-30°C water bath shaker; then centrifuge at 23-25°C and a relative centrifugal force of 15000-30000 g for 30-90 min, discard the precipitate to obtain supernatant E;

[0011] 7. Pipette the supernatant E into a dialysis bag, stir at 1-6°C for 20-50h, replace the liquid every 6h, take it out and concentrate it with polyethylene glycol to 1 / 2 of the original volume to obtain animal digestive tract mucin.

[0012] In the extraction and preparation of the crude mucus extract of the present invention, adding PBS with a volume of 5 times the sample to the scraped mucus can greatly shorten the experimental time; using 2×CPI-MEB (the volume ratio of cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB is 2:1) for further extraction can better ensure the freshness and activity of the extracted mucin and improve the extraction efficiency. The present invention prolongs the incubation time of cysteine ​​protease inhibitors, and then guarantees the freshness and activity of the extracted protein as much as possible under the condition of reducing the concentration gradient of cysteine ​​protease inhibitors, and can also achieve the effect consistent with the protein extraction concentration. The centrifugal speed is reduced during the extraction process of the present invention, so the difficulty of extraction is reduced in terms of instrument selection and operability. At the same time, the extract is screened and the dosage is explored and optimized, the dialysis box is changed to a dialysis bag, the concentration is changed from an ultrafiltration tube to a polyethylene glycol concentration, the amount of cysteine ​​protease inhibitor required is reduced by half, and the cost of extraction is greatly reduced. The method of the invention shortens the experimental time, ensures the freshness of the protein, and the electrophoresis detection result shows no tailing phenomenon, has good extraction effect and high extraction purity, and solves the problem that the existing method for extracting and purifying animal digestive tract mucin cannot achieve the best effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The electrophoresis diagram of digestive tract mucin extracted by the method of the present invention in Example 1;

[0014] Figure 2 The electrophoresis diagram of digestive tract mucin extracted by the control method in Example 1; DETAILED DESCRIPTION

[0015] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0017] Specific embodiment 1: The method for extracting and purifying animal digestive tract mucin in this embodiment is carried out according to the following steps:

[0018] 1. Obtaining mucus samples: When sampling, rinse the intestinal segment with phosphate buffered saline (PBS), gently scrape the intestinal segment to obtain mucus and phosphate buffered saline (PBS) at a weight ratio of 1:5, mix them by centrifugation to retain the precipitate, and then add 5 times the volume of phosphate buffered saline (PBS) to the precipitate to obtain the mucus sample;

[0019] 2. Add 5 times the volume of the cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB mixed solution to the mucus sample obtained in step 1 and mix evenly, the volume ratio of cysteine ​​protease inhibitor CPI to membrane extraction buffer MEB is 2:1, and incubate overnight at 2-6°C to obtain sample A;

[0020] 3. Sample A was placed in a centrifuge tube, centrifuged for 10-80 min at a temperature of 0-6°C and a relative centrifugal force of 15000-30000g, and the supernatant was discarded; 5 times the volume of a mixture of cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB was added and mixed evenly, the volume ratio of cysteine ​​protease inhibitor CPI to membrane extraction buffer MEB was 2:1, and centrifuged at a temperature of 0-6°C and a relative centrifugal force of 15000-30000g, and the supernatant was discarded to obtain precipitate B;

[0021] 4. The precipitate B obtained in step 3 is placed in a centrifuge tube and centrifuged for 10-80 minutes at a temperature of 0-6°C and a relative centrifugal force of 15000-30000g, and the supernatant is discarded; 5 volumes of a mixture of cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB are added and mixed evenly, the volume ratio of cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB is 2:1, and the supernatant is discarded and the precipitate is retained by centrifugation at a temperature of 0-6°C and a relative centrifugal force of 15000-30000g. The precipitate repeats the process of centrifugation described in this step, adding CPI and MEB mixture and centrifuging again 1-2 times to obtain precipitate C;

[0022] 5. Fill the centrifuge tube containing the precipitate C with membrane extraction buffer MEB, add 50 μL of 2 mol / L dithiothreitol DTT to each tube per 1 mL of membrane extraction buffer MEB, fill to a final concentration of 100 mmol / L dithiothreitol DTT, and incubate overnight on a water bath shaker at 30-40°C; add 50 μL of 2 mol / L dithiothreitol DTT to each 1 mL of membrane extraction buffer MEB, and incubate on a water bath shaker at 30-40°C for 5-15 hours to obtain sample solution D;

[0023] 6. Add 300 μL 1.5 mol / L iodoacetamide IAA to every 1.5 mL sample solution D, store in dark, and incubate overnight in a 20-30°C water bath shaker; then centrifuge at 23-25°C and a relative centrifugal force of 15000-30000 g for 30-90 min, discard the precipitate to obtain supernatant E;

[0024] 7. Pipette the supernatant E into a dialysis bag, stir at 1-6°C for 20-50h, replace the liquid every 6h, take it out and concentrate it with polyethylene glycol to 1 / 2 of the original volume to obtain animal digestive tract mucin.

[0025] In this embodiment, phosphate buffer (PBS) includes 10 mmol / L (a mixture of 13.7 mmol / L sodium chloride NaCl, 0.27 mmol / L potassium chloride KCl and 1 mmol / L disodium phosphate (Na2HPO4)), 0.18 mmol / L dipotassium phosphate (KH2PO4), and the cysteine ​​protease inhibitor CPI is purchased from the market.

[0026] In step 1 of this embodiment, the intestinal segment is gently scraped with a glass slide to obtain a mucus sample.

[0027] In step 2 of this embodiment, 2×CPI-MEB with a volume 5 times that of the sample is added, and the sample and the buffer are thoroughly mixed using a handheld automatic homogenizer.

[0028] In step 4 of this embodiment, the centrifuge tube is repeatedly centrifuged until the cysteine ​​protease inhibitor CPI solution is reduced to 1× (the required amount of cysteine ​​protease inhibitor varies according to the sample volume, and 1× means 1 times the concentration).

[0029] In step five of this embodiment, 50 μL of 2 mol / L DTT is added to make its concentration reach 200 mmol / L.

[0030] In step six of this embodiment, 300 μL of 1.5 mol / L IAA is added to every 1.5 mL of sample solution D, so that the final concentration of IAA is 250 mmol / L.

[0031] This embodiment obtains the identification of animal digestive tract mucin:

[0032] In order to determine the type of mucin, SDS-PAGE electrophoresis combined with mass spectrometry (QE) was used for identification. The specific operation of SDS-PAGE electrophoresis is as follows:

[0033] Put the thin plate and the thick plate together, with the upper edge facing up, and clamp them after balancing;

[0034] Add water regularly to check for leakage;

[0035] Add an equal volume of 10% precast gel bottom layer solution and bottom layer buffer solution and mix well, then continue to add the modified coagulant;

[0036] Pour the above mixed solution into the glass plate for glue making, and then add appropriate amount of ethanol to seal. After the lower layer of glue solidifies, pour out the ethanol and absorb it with filter paper;

[0037] Take equal volumes of upper gel solution and colored upper gel buffer and mix them evenly, then add the modified coagulant, inject into the glass plate to fill it up, and insert a 10-well lane comb;

[0038] After SDS-PAGE electrophoresis, the entire gel lane was cut out and placed in a clean 15 mL centrifuge tube, which was filled with pre-cooled double distilled water to prevent the gel from drying up. The data obtained by mass spectrometry detection were searched in the LC-MS / MS database using MASCOT software.

[0039] In this embodiment, adding 5 times the volume of dilution buffer shortens the test time, and in the centrifugation in step 4, the extraction time is further shortened. After shortening the extraction time, this embodiment can ensure the freshness and activity of the extracted mucin as much as possible, thereby improving the extraction efficiency. This embodiment reduces the speed of centrifugation, thereby reducing the difficulty of extraction in terms of instrument selection and operability. In this embodiment, the amount of cysteine ​​protease inhibitor required is reduced by half, the dialysis box is changed to a dialysis bag, and the concentration is changed from an ultrafiltration tube to a polyethylene glycol concentration, which greatly reduces the cost of extraction.

[0040] Specific implementation method 2: This implementation method is different from specific implementation method 1 in that: the centrifugal conditions in step 1 are: centrifugation for 15-50 min at a temperature of 0-6°C and a relative centrifugal force of 13000-30000g. The other steps and parameters are the same as those in specific implementation method 1.

[0041] Specific embodiment 3: This embodiment is different from the specific embodiment 1 in that: in the method for extracting and purifying animal digestive tract mucin, the membrane extraction buffer MEB: 6 mol / L guanidine chloride (GuCl), 0.1 mol / L Tris-HCl at pH 8.0, 5 mmol / L disodium ethylenediaminetetraacetate (EDTA2Na); dithiothreitol (DTT) is obtained by diluting DTT with the membrane extraction buffer MEB to a concentration of 2 mol / L; iodoacetamide IAA is obtained by diluting the membrane extraction buffer MEB to a concentration of 1.5 mol / L. The other steps and parameters are the same as those in the specific embodiment 1.

[0042] Example 1 Extraction of digestive tract mucin from weaned piglets using the method of the present invention in April 2023

[0043] (I) Solution preparation:

[0044] (1) Membrane extraction buffer (MEB): 6 M (mol / L) guanidine chloride (GuCl), 0.1 M (mol / L) Tris-HCl (pH 8.0), 5 mM (mmol / L) disodium ethylenediaminetetraacetate (EDTA2Na);

[0045] (2) Phosphate buffered saline (PBS): 10 mM (mmol / L) (13.7 mM (mmol / L) sodium chloride (NaCl) 0.27 mM (mmol / L) potassium chloride (KCl), 1 mM (mmol / L) disodium phosphate (Na2HPO4), 0.18 mM (mmol / L) dipotassium phosphate (KH2PO4);

[0046] ⑶ Dithiothreitol (DTT): 2MDTT in MEB, that is, dithiothreitol DTT was diluted with membrane extraction buffer MEB, and the concentration of dithiothreitol was 2 mol / L;

[0047] (4) Iodoacetamide (IAA): 1.5 mol / L IAA in MEB, i.e., dilute iodoacetamide with membrane extraction buffer MEB, and the concentration of iodoacetamide IAA is 1.5 mol / L;

[0048] ⑸CPI is a cysteine ​​protease inhibitor;

[0049] (II) Extraction and preparation of crude mucus extract: when sampling, rinse the intestinal segment with phosphate buffer solution (PBS), gently scrape the intestinal segment to obtain mucus and phosphate buffer solution (PBS) in a weight ratio of 1:5, mix them by centrifugation, and then add 5 times the volume of phosphate buffer solution (PBS) to the precipitate to obtain a mucus sample; that is, add 5 times the volume of the sample PBS to the scraped mucus and mix well; centrifuge at 13000-30000×g at 0-6℃ for 15-50min; after centrifugation, put the supernatant and precipitate into different centrifuge tubes respectively, add 5 times the volume of PBS to the precipitate, and mix well; take the required amount of supernatant and precipitate for backup, and put the rest into cryopreservation tubes and store at -80℃;

[0050] (III) Establishment of mucin extraction and purification method

[0051] Add 2×CPI-MEB (2×CPI-MEB: CPI and MEB mixture were mixed evenly, and the volume ratio of CPI and MEB was 2:1) with 5 times the volume of the mucus sample;

[0052] 2. Use a handheld automatic homogenizer to thoroughly mix the sample and buffer;

[0053] 3. Place the prepared test tube on a rotator and incubate overnight at 2-6°C;

[0054] 4. Remove the test tube from the rotator, transfer it to a centrifuge tube, centrifuge at 0-6°C, 15000-30000×g for 10-80 min, and discard the supernatant;

[0055] 5. Add 5 times 2×CPI-MEB buffer to the precipitate again (2×CPI-MEB: CPI and MEB mixed solution are mixed evenly, and the volume ratio of CPI and MEB is 2:1);

[0056] 6. Place the test tube on a rotator and run at 0-10°C for 3-10 hours;

[0057] 7. Repeat steps 4-61-3 times to complete the extraction, reducing the cysteine ​​protease inhibitor solution to 1×;

[0058] 8. After completion, fill the tubes with MEB and add 50 μL 2M DTT per 1 mL MEB in each tube (final DTT concentration is 100 mM);

[0059] 9. Place the test tube in a water bath shaker and incubate overnight at 30-40°C;

[0060] 10. Add 50 μL of 2M DTT to a concentration of 200 mM; return the tube to the water bath shaker and incubate at 30-40°C for 5-15 hours;

[0061] 11. Add 300 μL 1.5M IAA per 1.5 mL sample solution (final concentration of 1AA / MEB 250 mM);

[0062] 12. After adding IAA, the tube must be kept in the dark; place the tube back in the water bath shaker and incubate overnight at room temperature (20-30°C) (wrap the tube with tin foil);

[0063] 13. Spin the tube at 15,000-30,000 × g for 30-90 min at room temperature (10-25°C);

[0064] 14. Discard the precipitate and transfer the remaining solution directly to the dialysis bag and stir slowly at 1-6℃ for 20-50h, changing the liquid every 6h;

[0065] 15. After taking out, concentrate with polyethylene glycol to 1 / 2 of the original volume;

[0066] (III) Identification of mucin

[0067] In order to determine the type of mucin, SDS-PAGE electrophoresis combined with mass spectrometry (QE) was used for identification. The specific operation of SDS-PAGE electrophoresis is as follows:

[0068] 1. Put the thin plate and the thick plate together, punch the top edge upwards, and clamp them after balancing;

[0069] 2. Add water to check for leakage;

[0070] 3. Add an equal volume of 10% precast gel bottom layer solution and bottom layer buffer solution, mix well, and continue to add the improved coagulant;

[0071] 4. Pour the above mixed solution into the glass plate for glue making, and then add appropriate amount of ethanol to seal. After the lower layer of glue solidifies, pour out the ethanol and absorb it with filter paper;

[0072] 5. Take equal volumes of upper gel solution and colored upper gel buffer and mix them evenly, then add the improved coagulant, inject into the glass plate to fill it up, and insert a 10-hole swimming lane comb;

[0073] 6. After SDS-PAGE electrophoresis, cut out the entire gel lane and place it in a clean 15 mL centrifuge tube, which is filled with pre-cooled double distilled water to prevent the gel from drying up. The data obtained by mass spectrometry detection are searched in the LC-MS / MS database using MASCOT software.

[0074] (IV) Identification of mucin

[0075] The electrophoretic diagram after SDS-PAGE separation is as follows Figure 1 As shown in the figure, A. Marker; B. Extracted and purified mucin, the bands are clear and there is no protein degradation tailing. All the corresponding gel bands were cut off and placed in a clean centrifuge tube, filled with pre-cooled double distilled water to prevent the gel from drying up. The obtained protein was detected by mass (QE) spectrometry, and the data was searched in the LC-MS / MS database using MASCOT software. The identification results showed that there were three types of mucins in the ileal mucus of weaned piglets, MUC2, MUC7, and MUC13B (as shown in Table 1).

[0076] Table 1 Mass spectrometry identification of ileal mucin in weaned piglets

[0077]

[0078] The extraction of mucin was performed according to the method of Xia Bing (2022). The extraction and separation steps of mucin in the method of Xia Bing (2022) are as follows:

[0079] 1. Take an appropriate amount of collected mucosal samples, add 5 times the volume of extraction buffer (6M GuHCl, 0.1M Tris-HCl, 1mM EDTA, 0.1mM PMSF, 5mM NEM, pH 8.0) and homogenize, and shake overnight at 4°C and 180rpm;

[0080] 2. Centrifuge at 20,000 g for 1 h, collect the supernatant, add extraction buffer to the precipitate again, and shake overnight;

[0081] 3. Repeat step (2) once, and finally combine all supernatants; (4) Add DTT to a final concentration of 100 mM and perform reduction in a 37°C water bath;

[0082] 4. The next day, add IAA at a final concentration of 250 mM and allow to alkylate for 4 hours;

[0083] 5. The obtained solution was dialyzed with ultrapure water for 3 days, and the ultrapure water was replaced every 12 hours;

[0084] 6. After dialysis, freeze dry the product using a freeze dryer.

[0085] The results of mucin extraction using Xia Bing (2022) method are as follows Figure 2 As shown, from Figure 2 It can be seen that the extracted mucin was degraded, the bands were severely tailed, and no mucin expression was detected by mass spectrometry analysis.

[0086] pass Figure 1 and Figure 2 It can be seen that the mucin extracted by the present invention has good effect and high purity, and the present invention solves the problem that the existing methods for extracting and purifying animal digestive tract mucin cannot achieve the best effect.

Claims

1. A method for extracting and purifying mucin from the digestive tract of weaned piglets, characterized in that The method for extracting and purifying mucin from the digestive tract of weaned piglets is carried out according to the following steps:

1. Obtaining mucus samples: When sampling, rinse the intestinal segment with phosphate buffered saline (PBS), gently scrape the intestinal segment to obtain mucus and phosphate buffered saline (PBS) at a weight ratio of 1:5, mix them by centrifugation to retain the precipitate, and then add 5 times the volume of phosphate buffered saline (PBS) to the precipitate to obtain the mucus sample; 2. Add 5 times the volume of the cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB mixed solution to the mucus sample obtained in step 1 and mix evenly, the volume ratio of cysteine ​​protease inhibitor CPI to membrane extraction buffer MEB is 2:1, and incubate overnight at 2-6°C to obtain sample A; 3. Sample A was placed in a centrifuge tube, centrifuged for 10-80 min at a temperature of 0-6°C and a relative centrifugal force of 15000-30000 g, and the supernatant was discarded; 5 times the volume of a mixture of cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB was added and mixed evenly, the volume ratio of cysteine ​​protease inhibitor CPI to membrane extraction buffer MEB was 2:1, and centrifuged at a temperature of 0-6°C and a relative centrifugal force of 15000-30000 g to discard the supernatant and retain the precipitate to obtain precipitate B; 4. The precipitate B obtained in step 3 is placed in a centrifuge tube and centrifuged for 10-80 min at a temperature of 0-6°C and a relative centrifugal force of 15000-30000 g, and the supernatant is discarded; 5 times the volume of a mixture of cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB is added and mixed evenly, the volume ratio of cysteine ​​protease inhibitor CPI and membrane extraction buffer MEB is 2:1, and the supernatant is discarded and the precipitate is retained at a temperature of 0-6°C and a relative centrifugal force of 15000-30000 g. The precipitate repeats the process of centrifugation described in this step, adding CPI and MEB mixture and centrifuging again 1-2 times to obtain precipitate C; 5. Fill the centrifuge tube containing the precipitate C with membrane extraction buffer MEB, add 50 μL of 2 mol / L dithiothreitol DTT to each 1 mL of membrane extraction buffer MEB in each tube, and incubate overnight on a water bath shaker at 30-40°C; add 50 μL of 2 mol / L dithiothreitol DTT to each 1 mL of membrane extraction buffer MEB, and incubate on a water bath shaker at 30-40°C for 5-15 hours to obtain sample solution D; 6. Add 300 μL 1.5 mol / L iodoacetamide IAA to every 1.5 mL sample solution D, store in dark, and incubate overnight in a 20-30°C water bath shaker; then centrifuge at 23-25°C and a relative centrifugal force of 15000-30000g for 30-90 min, discard the precipitate and obtain the supernatant E; 7. Pipette the supernatant E into a dialysis bag, stir at 1-6°C for 20-50 h, replace the liquid every 6 h, take it out and concentrate it with polyethylene glycol to 1 / 2 of the original volume to obtain the weaned piglet digestive tract mucin; wherein the membrane extraction buffer MEB comprises: 6 mol / L guanidine chloride, 0.1 mol / L Tris-HCl at pH 8.0, and 5 mmol / L disodium ethylenediaminetetraacetic acid EDTA2Na.

2. The method for extracting and purifying mucin from the digestive tract of weaned piglets according to claim 1, characterized in that Centrifugation conditions in step 1: centrifuge at a temperature of 0-6°C and a relative centrifugal force of 13000-30000g for 15-50 min.

3. The method for extracting and purifying mucin from the digestive tract of weaned piglets according to claim 1, characterized in that In the method for extracting and purifying weaned piglet digestive tract mucin, membrane extraction buffer MEB includes: 6 mol / L guanidine chloride, 0.1 mol / L Tris-HCl at pH 8.0, and 5 mmol / L disodium ethylenediaminetetraacetic acid EDTA2Na; dithiothreitol DTT is obtained by diluting DTT with membrane extraction buffer MEB to a concentration of 2 mol / L; iodoacetamide IAA is obtained by diluting iodoacetamide with membrane extraction buffer MEB to a concentration of 1.5 mol / L.

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