Plant exosome-like vesicle wrapping egg yolk antibody as well as preparation method and application of plant exosome-like vesicle

Abstract

The invention discloses a preparation method of plant exosome-like vesicles wrapping egg yolk antibodies, which comprises the following steps: redissolving stably modified plant exosome-like vesicles to obtain redissolved plant exosome-like vesicles; freezing the re-dissolved plant source exosome sample vesicles, so as to obtain a frozen plant source exosome sample vesicle; repeating the operations of S1 and S2 for multiple times, and re-dissolving the plant-derived exosome-like vesicles again; taking an egg yolk antibody, and ultrasonically dissolving the egg yolk antibody with a PBS buffer solution for later use to obtain an egg yolk antibody solution; mixing the egg yolk antibody solution with the re-dissolved plant-derived exosome-like vesicles, and incubating to obtain the plant exosome-like vesicles coated with the egg yolk antibody; and redissolving the plant exosome-like vesicles wrapped with the egg yolk antibodies, performing vacuum freeze-drying, and preserving for later use to prepare the plant exosome-like vesicles wrapped with the egg yolk antibodies. The invention is used for solving the technical problem of more helicobacter pylori infection.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a plant exosome-like vesicle encapsulating an egg yolk antibody, and a preparation method and application thereof. Background Art

[0002] Helicobacter pylori (H. pylori) is a Gram-negative bacillus that primarily colonizes the gastric antral mucosa and adheres to gastric epithelial cells, resulting in a high infection rate. In my country, the H. pylori infection rate exceeds 50%, with a household infection rate exceeding 70%, affecting over 800 million people. At least 90 million people require treatment annually. H. pylori infection is a major cause of gastritis, gastric ulcers, duodenal ulcers, and gastric cancer, and is designated a Class 1 carcinogen by the World Health Organization. Over 95% of gastric cancer deaths are attributable to H. pylori infection. Preventing and treating H. pylori infection is key to reducing the incidence of gastric cancer.

[0003] Currently, the primary treatment for Helicobacter pylori is a quadruple antibiotic regimen. However, this approach has drawbacks, including antibiotic resistance, poor patient tolerance, disruption of gastrointestinal flora balance, and a high recurrence rate. It is not recommended for children under 14 years old. Therefore, developing new methods for the prevention and treatment of Helicobacter pylori infection, especially for adolescents, is crucial.

[0004] Yolk antibody (IgY) refers to a specific antibody protein produced by chickens through passive immunization. Numerous studies have demonstrated that specific anti-Helicobacter pylori antibody proteins are effective in treating H. pylori infection and are not prone to developing drug resistance. However, after oral administration of IgY, strong acid and pepsin disrupt the binding site of the specific immunoglobulin, reducing or even inactivating the IgY. Consequently, it is difficult for IgY to bind to H. pylori in the gastrointestinal tract, thereby preventing and treating H. pylori infection.

[0005] Plant exosome-like nanovesicles are small nanoscale vesicles secreted by plant cells. They typically range in diameter from 30 to 300 nm and have a lipid bilayer structure. They are widely available and contain a rich supply of bioactive substances. They have attracted considerable attention as potential nano-drug delivery systems due to their excellent biocompatibility, stability, and lack of toxic side effects. Research has shown that plant exosome-like nanovesicles can serve as natural transporters for a variety of molecules, bioactive substances, and a range of compounds during physiological processes.

[0006] Therefore, the development and utilization of plant exosome nano-like vesicles and the introduction of egg yolk antibody nanoparticles into them for encapsulation and modification can not only ensure the activity of egg yolk antibody IgY and specifically target the stomach against Helicobacter pylori, but also relieve stomach discomfort and repair gastric mucosa, which is of important and practical significance. Summary of the Invention

[0007] The present invention provides a plant exosome-like vesicle encapsulating an egg yolk antibody, a preparation method thereof, and an application thereof, to solve the current technical problem of multiple Helicobacter pylori infections.

[0008] In view of this, the present invention provides a method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies, comprising the following steps:

[0009] S1: re-dissolving the stably modified plant-derived exosome-like vesicles to obtain re-dissolved plant-derived exosome-like vesicles;

[0010] S2: Freeze the reconstituted plant-derived exosome-like vesicles;

[0011] S3: Repeat S1 and S2 steps multiple times to redissolve the plant-derived exosome-like vesicles;

[0012] S4: Take the egg yolk antibody, dissolve it with PBS buffer by ultrasonication and set aside to obtain the egg yolk antibody solution;

[0013] S5: mixing the egg yolk antibody solution with the re-dissolved plant-derived exosome-like vesicles, and incubating the mixture to obtain plant-derived exosome-like vesicles encapsulating the egg yolk antibody;

[0014] S6: The plant exosome-like vesicles encapsulating the egg yolk antibody are vacuum-lyophilized and stored for later use, thereby obtaining the plant exosome-like vesicles encapsulating the egg yolk antibody.

[0015] Optionally, the volume ratio of the egg yolk antibody to the stably modified plant-derived exosome-like vesicles is 1:(10-50).

[0016] Optionally, the incubation temperature in step S5 is 0-4°C, the incubation time is 12-36 hours, and the shaking speed is 50-120 rpm.

[0017] Optionally, the re-dissolution temperature of the plant exosome-like vesicles in steps S1, S3, and S6 is 0-4°C, the freezing condition in step S2 is -70-90°C, and the pre-freezing temperature of the vacuum freeze-drying in step S6 is -18-

[0018] (-20)℃, freeze-drying temperature is -38-(-40)℃, and freeze-drying time is 36-48h.

[0019] Optionally, the stably modified plant-derived exosome-like vesicles are prepared by the following method:

[0020] A1: Wash the plant source, add buffer, crush and extract the juice to obtain a plant source homogenate;

[0021] A2: Add enzymes to the plant source homogenate for enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0022] A3: Filter the enzymatic hydrolyzate to obtain a plant-derived filtrate;

[0023] A4: Centrifuge the plant-derived filtrate to obtain a crude exosome suspension;

[0024] A5: Purify the crude exosome resuspension and centrifuge to obtain a purified plant exosome-like vesicle solution;

[0025] A6: Mix the purified plant exosome-like vesicles with fatty acids, cholesterol, and PBS buffer to obtain preliminarily modified plant-derived exosome-like vesicles;

[0026] A7: The primary modified plant-derived exosome-like vesicles are mixed with PEG800 to obtain secondary modified plant-derived exosome-like vesicles;

[0027] A8: Add the secondary modified plant-derived exosome-like vesicles to the amino acid solution, centrifuge and resuspend to obtain the stably modified plant-derived exosome-like vesicles, which are then stored for future use.

[0028] Furthermore, the stably modified plant-derived exosome-like vesicles are prepared by the following method:

[0029] A1: Clean the plant source to remove surface stains, rinse with purified water 3-5 times, then rinse with distilled water 3-5 times, add buffer, crush and extract juice to obtain a plant source homogenate;

[0030] A2: Add enzymes to the plant source homogenate for enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0031] A3: Filter the enzymatic hydrolyzate to obtain a plant-derived filtrate;

[0032] A4: Centrifuge the plant-derived filtrate to obtain a crude exosome suspension;

[0033] A5: Purify the crude exosome resuspension and centrifuge to obtain a purified plant exosome-like vesicle solution;

[0034] A6: Mix the purified plant exosome-like vesicles with fatty acids, cholesterol, and PBS buffer to obtain preliminarily modified plant-derived exosome-like vesicles;

[0035] A7: The primary modified plant-derived exosome-like vesicles are mixed with PEG800 to obtain secondary modified plant-derived exosome-like vesicles;

[0036] A8: Add the secondary modified plant-derived exosome-like vesicles to an amino acid solution, centrifuge, and resuspend to obtain stably modified plant-derived exosome-like vesicles, which are then stored for future use;

[0037] Among them, the plant source in step A1 includes one or more of Bletilla striata, Buddha's hand, and Aloe vera; the buffer in step A1 is any one of PBS buffer, Tris-HCl, HEPES, bicarbonate buffer, MES and other commonly used buffers for plant cell extraction; the enzyme in step A2 is one or more of cellulase, pectinase, and hemicellulase; the concentration of fatty acid / cholesterol in step A6 is 1-10mM, preferably 5nM, and the volume ratio of plant exosome-like vesicle purification solution, fatty acid, and cholesterol is 1:1:(5-15), preferably 1:1:10; PBS buffer The solution is the sum of the total volume of the mixed solutions of the first three, the mixing temperature is 20-25° C., and the time is 8-12 hours; the plant-derived exosome-like vesicle system preliminarily modified in step A7 is mixed with PEG800, the final concentration of PEG800 is 0.5-5%, and the modification time is 2-4 hours, preferably 3 hours; the amino acid in step A8 is one or more of proline, arginine, histidine, glycine, lysine, hydroxyproline, and aspartic acid; the concentration of the amino acid solution is 10-50 nM; the stably modified plant-derived exosome-like vesicles in step A8 can also be vacuum freeze-dried to prepare a lyophilized powder.

[0038] Optionally, the pH value of the buffer solution in step A1 is 5.8-7.5, and the weight ratio of the buffer solution to the plant source is (1-2):1.

[0039] Optionally, the amount of the enzyme added is 1-5 wt % of the plant source homogenate, the enzymatic hydrolysis temperature in step A2 is 40-52° C., and the enzymatic hydrolysis time is 30-120 min.

[0040] Optionally, the centrifugation in step A4 is carried out at a speed of 3000-5000g, a temperature of 0-4°C, and a centrifugation for 20-30 min to obtain a supernatant; a speed of 5000-7000g, a temperature of 0-4°C, and a centrifugation for 30-45 min to obtain a supernatant; a speed of 10000-12000g, a temperature of 0-4°C, and a centrifugation for 30-60 min to obtain a supernatant; and then a speed of 50000-100000g, a temperature of 0-4°C, and a centrifugation for 60 min to obtain a precipitate, which is resuspended in 1xPBS buffer.

[0041] Optionally, in step A5, the centrifugation speed is 10000-12000g, the temperature is 0-4°C, and the centrifugation is performed for 3-5 minutes; in step A8, the centrifugation speed is 12000-15000g, and the cell is resuspended with amino acids.

[0042] Application of plant exosome-like vesicles encapsulating egg yolk antibodies in the development of anti-Helicobacter pylori drugs, health products, and foods.

[0043] Furthermore, the application of plant exosome-like vesicles encapsulating yolk antibodies in the development of anti-Helicobacter pylori drugs, health products, and food, including but not limited to the development of tablets, powders, capsules, pills, granules, etc., has the effect of stable, efficient, and targeted elimination of Helicobacter pylori.

[0044] It can be seen from the above technical solutions that the embodiments of the present invention have the following advantages:

[0045] 1. The present invention extracts exosome-like vesicles from Bletilla striata, Citron strychnine, etc., and modifies them multiple times, making them more stable and convenient for storage and transportation.

[0046] 2. The present invention introduces egg yolk antibodies into plant exosome-like vesicles, avoiding the problem of HP-IgY being affected by gastric acid and pepsin after reaching the stomach and rapidly reducing its activity, and can target and efficiently exert its effect in the stomach.

[0047] 3. The present invention uses plant exosome-like vesicles to encapsulate the yolk antibody HP-IgY, avoiding the residual organic solvents caused by traditional encapsulation methods, and the product is safer.

[0048] 4. The exosome-like vesicle-modified yolk antibody HP-IgY prepared by the present invention can target the stomach and bind to Helicobacter pylori, while relieving stomach discomfort and promoting gastric mucosal repair. It solves the problems of bacterial resistance and side effects caused by traditional quadruple antibiotics in the process of eradicating Helicobacter pylori, and destroying intestinal flora, providing new ideas for the treatment of Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0050] Figure 1 It is a flow chart of the present invention.

[0051] Figure 2 This is a scanning electron microscopy image of exosome-like vesicles from Bletilla striata.

[0052] Figure 3 This is a scanning electron micrograph of exosome-like vesicles from Bletilla striata encapsulated with yolk antibodies.

[0053] Figure 4 This is the result of the egg yolk antibody activity test in the application example.

[0054] Figure 5 This is the effect evaluation result of the application example.

[0055] Figure 6 is the C13 test result of patient 1;

[0056] Figure 7 This is the C13 test result of patient 2;

[0057] Figure 8 This is the C13 test result of patient 3. DETAILED DESCRIPTION

[0058] In order to make those skilled in the art better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or can be prepared by existing methods.

[0059] The egg yolk antibody was selected from Youlikang (Jiangsu) Biotechnology Co., Ltd. with a concentration of 10-25 mg / mL and an anti-Helicobacter pylori titer of not less than 1:160,000.

[0060] Preparation Example

[0061] Preparation Example 1

[0062] A stably modified plant-derived exosome-like vesicle is prepared by the following method:

[0063] A1: Wash the roots of Bletilla striata three times with purified water to remove surface stains, then wash five times with distilled water. Add 1x PBS buffer (pH 5.8) and extract the juice using a juicer to obtain a Bletilla striata root homogenate.

[0064] A2: Add 1% wt cellulase and 5% wt pectinase to the homogenate of Bletilla striata root, respectively, and perform enzymolysis in a 52°C water bath for 120 min to obtain an enzymatic hydrolyzate.

[0065] A3: Filter the enzymatic hydrolyzate through 4 layers of medical gauze to obtain the Bletilla striata root filtrate;

[0066] A4: Ultracentrifuge the filtrate from the root of Bletilla striata at 3000 g, 4°C, for 30 min to obtain the supernatant; at 5000 g, 4°C, for 45 min to obtain the supernatant; and at 12000 g, 4°C, for 30 min to obtain the supernatant. Centrifuge again at 100000 g, 4°C, for 60 min, collect the precipitate, and resuspend it in 1x PBS buffer to obtain a crude exosome suspension.

[0067] A5: Transfer the crude exosome resuspension to an exosome purification column and centrifuge at 10,000 g at 4°C for 5 min to obtain the purified exosome-like vesicles of Bletilla striata.

[0068] A6: Mix 10 parts of purified Bletilla striata exosome-like vesicles solution, 1 part of 5 mM fatty acid, and 1 part of cholesterol, then add 12 parts of PBS buffer and mix at 25°C for 12 hours to obtain preliminarily modified Bletilla striata exosome-like vesicles;

[0069] A7: The primary modified Bletilla striata exosome-like vesicles were mixed with PEG800 to a final concentration of 0.5%, and reacted at 25°C for 3 h to obtain secondary modified Bletilla striata exosome-like vesicles;

[0070] A8: Add the secondary modified Bletilla striata exosome-like vesicles to a glycine protection solution to a final concentration of 10 mM. Centrifuge at 15,000 g and resuspend in glycine to obtain stably modified Bletilla striata exosome-like vesicles. Store at -80°C for future use.

[0071] Preparation Example 2

[0072] A stably modified plant-derived exosome-like vesicle is prepared by the following method:

[0073] A1: Take the root of Bletilla striata, wash it four times with purified water to remove surface stains, then wash it four times with distilled water. Add 2x the volume of PBS buffer (pH 6.5), and extract the juice with a juicer to obtain a Bletilla striata root homogenate.

[0074] A2: Add 5% wt cellulase and 5% wt pectinase to the homogenate of Bletilla striata root, respectively, and perform enzymolysis in a 40°C water bath for 30 min to obtain an enzymatic hydrolyzate.

[0075] A3: Filter the enzymatic hydrolyzate through 4 layers of medical gauze to obtain the Bletilla striata root filtrate;

[0076] A4: Ultracentrifuge the filtrate from the root of Bletilla striata at 5000 g, 0°C, for 20 min to obtain the supernatant; at 7000 g, 0°C, for 30 min to obtain the supernatant; and at 10000 g, 0°C, for 60 min to obtain the supernatant. Centrifuge again at 80000 g, 0°C, for 60 min, collect the precipitate, and resuspend it in 1x PBS buffer to obtain a crude exosome suspension.

[0077] A5: Transfer the crude exosome resuspension to an exosome purification column and centrifuge at 12000 g at 0°C for 3 min to obtain the purified exosome-like vesicles of Bletilla striata.

[0078] A6: Mix 15 parts of purified Bletilla striata exosome-like vesicles solution, 1 part of 10 mM fatty acid, and 1 part of cholesterol, then add 17 parts of PBS buffer and mix at 25°C for 8 hours to obtain preliminarily modified Bletilla striata exosome-like vesicles;

[0079] A7: The primary modified Bletilla striata exosome-like vesicles were mixed with PEG800 to a final concentration of 5%, and reacted at 25°C for 4 h to obtain secondary modified Bletilla striata exosome-like vesicles;

[0080] A8: Add the secondary modified Bletilla striata exosome-like vesicles into glycine protective solution to a final concentration of 25 mM. Centrifuge at 12,000 g and resuspend in glycine to obtain stably modified Bletilla striata exosome-like vesicles. Store at -80°C for future use.

[0081] Preparation Example 3

[0082] A stably modified plant-derived exosome-like vesicle is prepared by the following method:

[0083] A1: Take the root of Bletilla striata, wash it five times with purified water to remove surface stains, then wash it three times with distilled water, add 1.5 times the pH 7.5 PBS buffer, and extract the juice with a juicer to obtain a Bletilla striata root homogenate;

[0084] A2: Add 5% wt cellulase and 2.5% wt pectinase to the homogenate of Bletilla striata root, respectively, and perform enzymolysis in a 45°C water bath for 60 min to obtain an enzymatic hydrolyzate.

[0085] A3: Filter the enzymatic hydrolyzate through 4 layers of medical gauze to obtain the Bletilla striata root filtrate;

[0086] A4: Ultracentrifuge the filtrate from the root of Bletilla striata at 4000 g, 2°C, for 25 min to obtain the supernatant; at 6000 g, 2°C, for 40 min to obtain the supernatant; and at 11000 g, 2°C, for 45 min to obtain the supernatant. Centrifuge again at 50000 g, 2°C, for 60 min, collect the precipitate, and resuspend it in 1x PBS buffer to obtain a crude exosome suspension.

[0087] A5: Transfer the crude exosome resuspension to an exosome purification column and centrifuge at 11000g at 2°C for 4 min to obtain the purified exosome-like vesicles of Bletilla striata.

[0088] A6: Mix 10 parts of purified Bletilla striata exosome-like vesicles solution, 2 parts of 1 mM fatty acid, and 2 parts of cholesterol, then add 14 parts of PBS buffer and mix at 20°C for 10 hours to obtain preliminarily modified Bletilla striata exosome-like vesicles;

[0089] A7: The primary modified Bletilla striata exosome-like vesicles were mixed with PEG800 to a final concentration of 2.5%, and reacted at 23°C for 2 h to obtain secondary modified Bletilla striata exosome-like vesicles;

[0090] A8: Add the secondary modified Bletilla striata exosome-like vesicles into glycine protective solution to a final concentration of 25 mM. Centrifuge at 12,000 g and resuspend in glycine to obtain stably modified Bletilla striata exosome-like vesicles. Store at -80°C for future use.

[0091] Example

[0092] Example 1

[0093] A method for preparing plant exosome-like vesicles encapsulating yolk antibodies, referring to Figure 1-Figure 3 , including the following steps:

[0094] S1: The stably modified Bletilla striata exosome-like vesicles prepared in Preparation Example 1 were redissolved at 4° C. to obtain redissolved Bletilla striata exosome-like vesicles;

[0095] S2: The reconstituted Bletilla striata exosome-like vesicles were frozen at -80°C for 6 h;

[0096] S3: After repeating S1 and S2 for 5 cycles, the Bletilla striata exosome-like vesicles were redissolved at 4°C;

[0097] S4: Prepare 10 mg / mL egg yolk antibody using PBS buffer, dissolve it by ultrasonication and set aside to obtain egg yolk antibody solution;

[0098] S5: 1 part of the egg yolk antibody solution and 10 parts of the re-dissolved Bletilla striata exosome-like vesicles were mixed by shaking and placed on a shaker. The mixture was incubated at 4°C for 24 hours with a shaker speed of 120 rpm to obtain Bletilla striata exosome-like vesicles containing egg yolk antibodies.

[0099] S6: The exosome-like vesicles of Bletilla striata encapsulating the egg yolk antibody were vacuum-lyophilized under the following conditions: pre-freezing temperature -18°C, freeze-drying temperature -38°C, and freeze-drying time 48 hours to obtain the exosome-like vesicles of Bletilla striata encapsulating the egg yolk antibody, which was named YL1.

[0100] Example 2

[0101] A method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies comprises the following steps:

[0102] S1: The stably modified Bletilla striata exosome-like vesicles prepared in Preparation Example 2 were redissolved at 4° C. to obtain redissolved Bletilla striata exosome-like vesicles;

[0103] S2: Freeze the reconstituted Bletilla striata exosome-like vesicles at -80°C for 8 h;

[0104] S3: After repeating S1 and S2 for 4 cycles, the Bletilla striata exosome-like vesicles were redissolved at 4°C;

[0105] S4: Prepare 25 mg / mL egg yolk antibody using PBS buffer, dissolve it by ultrasonication and set aside to obtain egg yolk antibody solution;

[0106] S5: 1 part of the egg yolk antibody solution and 50 parts of the re-dissolved Bletilla striata exosome-like vesicles were shaken and placed on a shaker. The mixture was incubated at 0°C for 12 h with a shaker speed of 50 rpm to obtain Bletilla striata exosome-like vesicles containing egg yolk antibodies.

[0107] S6: The exosome-like vesicles of Bletilla striata encapsulating the egg yolk antibody were vacuum-lyophilized under the following conditions: pre-freezing temperature -20°C, freeze-drying temperature -40°C, and freeze-drying time 36 hours to obtain the exosome-like vesicles of Bletilla striata encapsulating the egg yolk antibody, which was named YL2.

[0108] Example 3

[0109] A method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies comprises the following steps:

[0110] S1: The stably modified Bletilla striata exosome-like vesicles prepared in Preparation Example 3 were redissolved at 2°C to obtain redissolved Bletilla striata exosome-like vesicles;

[0111] S2: Freeze the reconstituted Bletilla striata exosome-like vesicles at -80°C for 7 h;

[0112] S3: After repeating S1 and S2 for 3 cycles, the Bletilla striata exosome-like vesicles were redissolved at 2°C;

[0113] S4: Prepare 15 mg / mL egg yolk antibody using PBS buffer, dissolve it by ultrasonication and set aside to obtain egg yolk antibody solution;

[0114] S5: 1 part of the egg yolk antibody solution and 25 parts of the re-dissolved Bletilla striata exosome-like vesicles were mixed by shaking and placed on a shaker. The mixture was incubated at 2°C for 36 hours with a shaker speed of 60 rpm to obtain Bletilla striata exosome-like vesicles containing egg yolk antibodies.

[0115] S6: The exosome-like vesicles of Bletilla striata encapsulating the egg yolk antibody were vacuum-lyophilized under the following conditions: pre-freezing temperature -20°C, freeze-drying temperature -40°C, and freeze-drying time 42 hours to obtain the exosome-like vesicles of Bletilla striata encapsulating the egg yolk antibody, which was named YL3.

[0116] Application Examples

[0117] Application Example 1

[0118] Take YL1 prepared in Example 1, add probiotics, oligofructose, oligoxylose, lactitol, orange juice powder and other auxiliary materials to prepare a solid beverage, 2 grams per bag, ensuring that the added amount of egg yolk antibody content is not less than 100 mg / bag, and name it YYL1.

[0119] Application Example 2

[0120] The difference between Application Example 2 and Application Example 1 is that the sources of the Bletilla striata exosome-like vesicles encapsulating the egg yolk antibody are different. YL2 prepared in Example 2 is used and named YYL2.

[0121] Application Example 3

[0122] The difference between Application Example 3 and Application Example 1 is that the sources of the Bletilla striata exosome-like vesicles encapsulating the egg yolk antibody are different. YL3 prepared in Example 3 is used and named YYL3.

[0123] Comparative Example

[0124] The difference between the comparative example and application example 2 is that the exosome-like vesicles of Bletilla striata encapsulating the yolk antibody are replaced with an equal amount of the yolk antibody and named DBL.

[0125] Performance testing

[0126] Activity detection

[0127] 1g of each of Application Examples 1-3 and the Comparative Example was placed in 20mL of simulated gastric fluid and assayed for antibody activity after immersion for 0, 0.5, 1, 1.5, and 2 hours, respectively. The activity of the theoretical concentration of egg yolk antibody (measured in PBS buffer at pH 6.8) was used as a comparison. Activity retention (%) = test solution OD450nm / theoretical OD450nm × 100%.

[0128] Antibody activity was detected using ELISA.

[0129] (1) Add the sample to the ELISA plate at 100 μL / well, incubate at 37°C for 40 min, and wash five times with PBST.

[0130] (2) Enzyme-labeled antibody: dilute HRP-goat anti-chicken IgY antibody 1:5000 with enzyme diluent, 100 μL / well, incubate at 37°C for 40 min, and wash 5 times with PBST.

[0131] (3) Color development: 50 μL / well solution A + 50 μL / well solution B for 5 min, 50 μL / well stop solution to terminate the reaction, measure the OD450 value and record the results as shown in Table 1. The results of the yolk antibody in vivo detection and the effect evaluation results of the application example are shown in Table 1. Figure 4 and Figure 5 shown.

[0132] Table 1 Yolk antibody activity rate %

[0133]

[0134] As can be seen from Table 1, compared with the comparative example, the antibody activity of the products of Application Examples 1-3 is generally improved, the sustained-release effect is obvious, and the activity lasts longer.

[0135] Clinical effect evaluation

[0136] (1) In order to demonstrate the therapeutic effect of the present invention on Helicobacter pylori, 36 Helicobacter pylori-infected patients with stomach discomfort were recruited for clinical observation and treatment. The patients were of both genders and aged between 20 and 70 years old.

[0137] (2) Judgment criteria

[0138] Patients with a C13 breath test value ≥4 were considered positive and included in the group.

[0139] (3) Treatment methods

[0140] The patient took the product of Application Example 2 of the present invention, taking the product of Comparative Example 1 as a control, orally, and the usage and dosage were: take twice a day, one bag each time, and 7 days as a course of treatment.

[0141] (4) Efficacy standards

[0142] Negative: After treatment, the C13 breath test value is ≤4.

[0143] Symptoms disappear or improve: After treatment, discomfort symptoms (bad breath, stomach acid, stomach pain, belching, acid reflux) disappear or improve.

[0144] Ineffective: After treatment, the discomfort symptoms did not improve and the Helicobacter pylori test was positive.

[0145] (5) The treatment results are shown in Table 2. Three patients were randomly selected for C13 testing. The test results are shown in Table 2. Figure 6-Figure 8 shown.

[0146] Table 2 Treatment results statistics

[0147]

[0148] As can be seen from Table 2, the treatment results of Application Example 2 are better than those of the comparative example, indicating that the egg yolk-encapsulated plant-derived exosome-like vesicles of the present invention are more capable of targeting the stomach and binding to Helicobacter pylori, while also relieving stomach discomfort and promoting the repair of gastric mucosa. This solves the problems of bacterial resistance and side effects caused by traditional quadruple antibiotics in the process of eradicating Helicobacter pylori, and destroying intestinal flora, providing new ideas for the treatment of Helicobacter pylori.

[0149] As described above, the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments can still be modified, or some of the technical features thereof can be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies, characterized in that: The steps include: S1: re-dissolving the stably modified plant-derived exosome-like vesicles to obtain re-dissolved plant-derived exosome-like vesicles; S2: Freeze the reconstituted plant-derived exosome-like vesicles; S3: Repeat S1 and S2 steps multiple times to redissolve the plant-derived exosome-like vesicles; S4: Take the egg yolk antibody, dissolve it with PBS buffer by ultrasonication and set aside to obtain the egg yolk antibody solution; S5: mixing the egg yolk antibody solution with the re-dissolved plant-derived exosome-like vesicles, and incubating the mixture to obtain plant-derived exosome-like vesicles encapsulating the egg yolk antibody; S6: The plant exosome-like vesicles encapsulating the egg yolk antibody are vacuum-lyophilized and stored for later use, thereby obtaining the plant exosome-like vesicles encapsulating the egg yolk antibody.

2. The method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies according to claim 1, characterized in that: The volume ratio of the yolk antibody to the stably modified plant-derived exosome-like vesicles is 1:(10-50).

3. The method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies according to claim 1, characterized in that: The incubation temperature in step S5 is 0-4°C, the incubation time is 12-36 hours, and the shaking speed is 50-120 rpm.

4. The method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies according to claim 1, characterized in that: The reconstitution temperature of the plant exosome-like vesicles in steps S1, S3, and S6 is 0-4°C, the freezing condition in step S2 is -70-90°C, the pre-freezing temperature of the vacuum freeze-drying in step S6 is -18-(-20)°C, the freeze-drying temperature is -38-(-40)°C, and the freeze-drying time is 36-48h.

5. The method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies according to claim 1, characterized in that: The stably modified plant-derived exosome-like vesicles are prepared by the following method: A1: Wash the plant source, add buffer, crush and extract the juice to obtain a plant source homogenate; A2: Add enzymes to the plant source homogenate for enzymatic hydrolysis to obtain an enzymatic hydrolyzate; A3: Filter the enzymatic hydrolyzate to obtain a plant-derived filtrate; A4: Centrifuge the plant-derived filtrate to obtain a crude exosome resuspension; A5: Purify the crude exosome resuspension and centrifuge to obtain a purified plant exosome-like vesicle solution; A6: Mix the purified plant exosome-like vesicles with fatty acids, cholesterol, and PBS buffer to obtain preliminarily modified plant-derived exosome-like vesicles; A7: The primary modified plant-derived exosome-like vesicles are mixed with PEG800 to obtain secondary modified plant-derived exosome-like vesicles; A8: Add the secondary modified plant-derived exosome-like vesicles to the amino acid solution, centrifuge and resuspend to obtain the stably modified plant-derived exosome-like vesicles, which are then stored for future use.

6. The method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies according to claim 5, characterized in that: The pH value of the buffer solution in step A1 is 5.8-7.5, and the weight ratio of the buffer solution to the plant source is (1-2):

1.

7. The method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies according to claim 5, characterized in that: The amount of the enzyme added is 1-5 wt % of the plant source homogenate. The enzymatic hydrolysis temperature in step A2 is 40-52° C. and the enzymatic hydrolysis time is 30-120 min.

8. The method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies according to claim 5, characterized in that: The centrifugation in step A4 is carried out at a speed of 3000-5000g, a temperature of 0-4°C, for 20-30 min to obtain a supernatant; a speed of 5000-7000g, a temperature of 0-4°C, for 30-45 min to obtain a supernatant; a speed of 10000-12000g, a temperature of 0-4°C, for 30-60 min to obtain a supernatant; The pellet was centrifuged at a speed of 50,000-100,000 g and a temperature of 0-4°C for 60 min, and the pellet was resuspended in 1x PBS buffer.

9. The method for preparing plant exosome-like vesicles encapsulating egg yolk antibodies according to claim 5, characterized in that: In step A5, centrifuge at a speed of 10,000-12,000 g, a temperature of 0-4° C., and centrifuge for 3-5 minutes; in step A8, centrifuge at a speed of 12,000-15,000 g and resuspend with amino acids.

10. Use of the plant exosome-like vesicles encapsulating egg yolk antibodies according to any one of claims 1 to 9 in the development of anti-Helicobacter pylori drugs, health products, and foods.

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