A preparation method for fish antigen presenting molecule MHCII positive exosomes

Abstract

The present invention discloses a method for preparing MHCII-positive exosomes rich in antigen presenting molecules, and belongs to the field of biotechnology. The method of the present invention comprises the following steps: after the Vibrio anguillarum is shaken to the logarithmic growth phase, it is inactivated; the inactivated Vibrio anguillarum solution is taken for intraperitoneal injection into fish, and the induction is 6-24h; after the fish is anesthetized, peripheral blood is extracted along the tail vein with a syringe; after the peripheral blood is removed, serum-free L15 culture medium is injected into the peritoneal cavity of the fish with a syringe; after massaging the fish abdomen, the peritoneal fluid is extracted along the upper edge of the abdominal cavity with a syringe; finally, the peritoneal fluid is separated by a continuous gradient centrifugation method to obtain MHCII-positive exosomes. The exosomes rich in antigen presenting molecules MHCII-positive prepared by the present invention have a significant effect in promoting T cell activation and vaccine immune efficacy. The present invention creatively establishes a method for preparing fish-rich antigen presenting molecules MHCII-positive exosomes with high efficiency, high purity and high yield through stimulants suitable for fish and a unique fish peritoneal lavage method, which has important scientific significance and application value.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of MHCII-positive exosomes rich in antigen presenting molecules, and belongs to the field of biotechnology. Background Art

[0002] Exosomes are small membrane vesicles (40-200nm) containing complex molecular patterns such as small RNA, messenger RNA and proteins. They are mainly derived from multivesicular bodies formed by the invagination of lysosomal microparticles. After the multivesicular outer membrane fuses with the cell membrane, it is released into the extracellular matrix. It has the ability to transfer "molecular cargo" to other cells and trigger physiological responses. Exosomes can be secreted by different types of cells and participate in many physiological functions and pathological processes, such as antigen presentation, cell activation, tumor growth and migration. However, there are few reports on fish exosomes, mainly because it is impossible to obtain a large number of high-purity exosomes to meet the needs of subsequent functional experiments.

[0003] Adequate initiation of an adaptive immune response depends on the presentation of pathogen-derived peptides in the context of major histocompatibility complex II (MHCII) on the surface of antigen-presenting cells, which are recognized by T cell receptors and activate CD4 + T cells, while secreting cytokines, further regulate the immune response. Studies have reported that exosomes have the potential to initiate specific immune responses. Exosomes can carry MHC class II molecules to directly or indirectly activate CD4 + T cells, thereby specifically inducing T cell immune responses, providing the possibility for T cell targeted drug delivery and targeted therapy.

[0004] However, it is difficult to isolate fish exosomes rich in MHCII-positive. There are two main problems: First, there is a lack of suitable sources for isolating exosomes: Currently, the sources of exosomes in vivo are mainly concentrated in urine, emulsion, blood, etc., but for fish, the exosomes isolated from these sources are usually small in quantity, and a large number of samples need to be collected to meet the demand. Second, there is a lack of suitable stimuli: inappropriate stimuli or insufficient stimulation intensity are not enough to cause fish to release exosomes rich in antigen-presenting molecules MHCII-positive, while excessive stimulation will cause fish peritoneal cells to release a large number of extracellular traps, causing cell aggregation, and the content of exosomes rich in antigen-presenting molecules MHCII-positive will also decrease. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide a method for quickly and inexpensively obtaining and isolating MHCII-positive exosomes rich in antigen presenting molecules, and obtaining high-purity and high-yield MHCII-positive exosomes through appropriate stimulation conditions and peritoneal lavage.

[0006] The present invention has found that fish peritoneal fluid contains abundant antigen presenting cells such as dendritic cells, macrophages, B cells, etc. A large number of antigen presenting cells are prone to produce natural exosomes rich in antigen presenting molecules. The peritoneal fluid is also more aggregated than other body fluids, providing a good source for exosome separation. However, there is currently no suitable method for separating fish peritoneal fluid, and there is no report on separating fish peritoneal exosomes. In addition, the present invention uses inactivated Vibrio anguillarum as a stimulus source, which is not infectious but immunogenic, and can effectively induce fish humoral immunity without causing excessive stress to the fish. Further, the obtained peritoneal fluid is separated by a continuous gradient centrifugation method, and finally fish exosomes rich in antigen presenting molecules MHCII positive are obtained.

[0007] The present invention comprises the following steps:

[0008] Step 1: inactivate Vibrio anguillarum by shaking it to the logarithmic growth phase;

[0009] Step 2: Take the inactivated Vibrio anguillarum solution and inject it into the fish intraperitoneally for 6-24 hours;

[0010] Step 3: After the fish is anesthetized, peripheral blood is drawn along the tail vein with a syringe; after the peripheral blood is removed, serum-free L15 culture medium is injected into the abdominal cavity of the fish with a syringe;

[0011] Step 4: After massaging the fish's abdomen, use a syringe to draw out the peritoneal fluid along the upper edge of the abdominal cavity;

[0012] Step 5: Separation of exosomes by continuous gradient centrifugation: o C, the peritoneal fluid was extracted and 500× g Centrifuge for 10 min, 4000× g Centrifuge for 30 min to remove cells and cell debris to obtain supernatant; cell supernatant 10000× g Centrifuge for 30 min to remove the large vesicles in the fish peritoneal cavity, and the cell supernatant is further heated at 115000× g Centrifuge for 90 min to obtain a precipitate; resuspend the precipitate in phosphate buffer and centrifuge at 115,000 × g After centrifugation for 90 min, the supernatant containing liposomes was removed, and the precipitate was resuspended in phosphate buffer to separate the positive exosomes rich in fish antigen presenting molecule MHCII.

[0013] The inactivation method of Vibrio anguillarum is to use formaldehyde solution, 37 o C, 180 rpm to inactivate it.

[0014] The fish is flounder.

[0015] The positive exosomes rich in fish antigen presenting molecule MHCII are used to induce CD4 + Application of T lymphocyte activation.

[0016] The positive exosomes rich in fish antigen presenting molecule MHCII are used to promote the immune efficacy of fish vaccines.

[0017] The advantages of the present invention are: the present invention creatively establishes a method for preparing fish antigen presenting molecule MHCII positive exosomes rich in high efficiency, high purity and high yield through stimulants suitable for fish and a unique fish peritoneal lavage method. The exosomes can participate in T cell activation and enhance the body's immune response. Compared with extraction from other parts, a large number of exosomes can be separated from the peritoneal cavity, the extraction efficiency is high, and the prepared exosomes have good integrity and biological activity. The exosomes separated after inactivated bacteria stimulate the peritoneal cavity can be rich in high concentrations of MHCII positive exosomes, have the function of specifically inducing T cell activation, and can be used in the preparation of vaccine promoters. The present invention provides a low-cost, high-yield, high-quality technology for extracting fish exosomes, which solves the problem that it is difficult to obtain a large number of fish exosomes and cannot be studied. This is a technological breakthrough in the study of fish exosomes, and has important scientific significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a transmission electron micrograph of the isolated peritoneal exosomes of the Japanese flounder.

[0019] Figure 2 This is the particle size diagram of the peritoneal exosomes of the isolated flounder detected by nanoflow cytometry.

[0020] Figure 3 This is a map of marker molecules for Western blotting to identify the isolated peritoneal exosomes of Japanese flounder.

[0021] In the figure, M is the standard protein, 1 is the binding reaction band of CD9 rabbit polyclonal antibody and 27kDa protein of peritoneal exosomes of Japanese flounder, 2 is the binding reaction band of CD81 rabbit polyclonal antibody and 25kDa protein of peritoneal exosomes of Japanese flounder, 3 is the binding reaction band of TSG101 rabbit polyclonal antibody and 44kDa protein of peritoneal exosomes of Japanese flounder, and 4 is the binding reaction band of MHCII mouse polyclonal antibody and 27kDa protein of peritoneal exosomes of Japanese flounder.

[0022] Figure 4 This is a transmission electron micrograph of isolated blood exosomes from flounder.

[0023] Figure 5 This is the particle size diagram of the exosomes separated from the blood of the Japanese flounder detected by nanoflow cytometry.

[0024] Figure 6This is a picture of MHCII-positive exosomes in the peritoneal cavity of Japanese flounder produced by different stimulation sources detected by Western blotting.

[0025] In the figure, GAPDH is the internal reference protein; Control uses PBS as the stimulus source, and Immunization uses inactivated Vibrio anguillarum as the stimulus source.

[0026] Figure 7 In order to co-incubate the exosomes rich in antigen presenting molecules MHCII positive with leukocytes, CD4 + Flow cytometry analysis of T cells.

[0027] Figure 8 This is a flow cytometric analysis of the dynamic changes of lymphocytes in the spleen, head kidney, and peripheral blood after injecting exosomes rich in antigen presenting molecules MHCII and inactivated Vibrio anguillarum into fish. DETAILED DESCRIPTION

[0028] In order to explain the technical solution of the present invention more clearly, the present invention is further described below with reference to the accompanying drawings and through specific embodiments.

[0029] Example 1: Extraction and separation of exosomes rich in antigen presenting molecules MHCII positive

[0030] Step 1: Use LB medium to shake Vibrio anguillarum to the logarithmic growth phase, measure and adjust the bacterial concentration to 1x10 9 CFU / mL, add 2% formaldehyde solution to the final volume, 37 o C, inactivate it at 180 rpm; 48 h after inactivation, use LB solid medium for coating culture to detect whether the inactivation is successful.

[0031] Step 2: Take a healthy flounder of about 0.5 kg and inject 200 μL of 1x10 8 CFU / mL inactivated Vibrio anguillarum solution, induced for 6-24h.

[0032] Step 3: Anesthetize the flounder with the anesthetic MS-222, and draw peripheral blood along the caudal vein with a 10 mL syringe (injection needle marking: 0.8 × 28 mm). After removing the peripheral blood, use a 10 ml syringe with a 0.26 × 9 mm injection needle to inject serum-free L15 medium into the fish's abdominal cavity.

[0033] Step 4: After massaging the fish abdomen, use a 20mL syringe with a 1.2×38mm needle to draw out the peritoneal fluid along the upper edge of the abdominal cavity and place it on ice for later use;

[0034] Step 5, in 4 oC, the peritoneal fluid was extracted and 500× g Centrifuge for 10 min, 4000× g Centrifuge for 30 min to remove cells and cell debris to obtain supernatant; cell supernatant 10000× g Centrifuge for 30 min to remove the large vesicles in the fish peritoneal cavity, and the cell supernatant is further heated at 115000× g Centrifuge for 90 min to obtain a precipitate; resuspend the precipitate in phosphate buffer and centrifuge at 115,000 × g After centrifugation for 90 min, the supernatant containing liposomes was removed, and the precipitate was resuspended in phosphate buffer to separate exosomes rich in antigen presenting molecules MHCII-positive.

[0035] Example 2: Detection of MHCII-positive exosomes rich in antigen presenting molecules

[0036] Step 1: Drop the PBS solution containing exosomes separated above onto the carbon-coated film of the copper mesh and let it settle for 3-5 minutes. Remove the floating liquid with filter paper, stain with 3% phosphotungstic acid, and air-dry at room temperature for 20 minutes. The size and morphology of the separated exosomes were observed under a H600IV transmission electron microscope. The results showed that the exosomes showed a classic "cup-and-saucer" structure, which was either dispersed or aggregated, with a particle size of 40-200nm. (See Figure 1 ).

[0037] Step 2: Nano flow cytometer was used to detect the particle size and content of exosomes. The exosome samples were loaded after the instrument performance test was qualified with standard products. Note that gradient dilution was required to avoid clogging of the injection needle by the sample. 3 μL of purified peritoneal exosome suspension was analyzed by flow cytometer (Flow NanoAnalyzer). The results showed that the average particle size of peritoneal exosomes of Japanese flounder was 57.25 nm, and 40-150 nm exosomes accounted for 99.81% of the total number of particles (see Figure 2 ).

[0038] Step 3: Add lysis buffer RIPA to the separated exosome precipitate and lyse on ice for 30 min. After lysis, centrifuge at 12000 rpm for 30 min at 4°C to obtain total exosome protein. Add the exosome protein to the sample buffer containing sodium dodecyl sulfate in equal proportion and boil in boiling water for 5 min. Load the sample on a 15% gel and add 15µl of sample to each well. Under constant current conditions (30mA), electrophoresis is stopped when the bromophenol blue indicator reaches the bottom edge. Remove the gel. Cut a nitrocellulose membrane (pore size 0.22µm) of the same size as the electrophoresis gel and wet it with electrotransfer buffer (electrotransfer buffer: 25mmol / LTris-Base, 192mmol / L glycine, 20% methanol, pH 8.3) and place it on the gel after electrophoresis. Cut off a corner on the nitrocellulose membrane to mark the starting end of the sample sequence. Use a moistened filter paper as support and stick a second moistened filter paper on the other side of the gel sheet; follow the above placement order to form a sandwich of gel, nitrocellulose membrane and filter paper; place the "gel sandwich" in an electrophoresis tank filled with electrotransfer buffer, with the nitrocellulose membrane facing the anode; run the electrophoresis at a constant voltage of 30V for 90 minutes; after the transfer is complete, remove the nitrocellulose membrane. Place in a 3% bovine serum albumin solution at 37 o C block for 2h; add exosome marker molecule antibodies CD9 (1:1000), CD81 (1:1000), TSG101 (1:1000) and antigen presenting molecule MHCII (1:1000) to the nitrocellulose membrane and rotate slowly at 4℃ overnight; wash three times with PBST, 10min each time; add HRP-labeled goat anti-rabbit Ig antibody (1:40000 dilution) to the nitrocellulose membrane and shake slowly at 37℃ for 1h; wash three times with PBST, 10min each time; add ECL colorimetric solution on the nitrocellulose membrane and detect using a chemiluminometer (FUSION FX Spectra, VILBER). The results showed that the CD9 rabbit polyclonal antibody could react with the peritoneal exosomes of the Japanese flounder at about 27 kDa, the CD81 rabbit polyclonal antibody could react with the peritoneal exosomes of the Japanese flounder at about 25 kDa, the TSG101 rabbit polyclonal antibody could react with the peritoneal exosomes of the Japanese flounder at about 44 kDa, and the MHCII mouse polyclonal antibody could react with the peritoneal exosomes of the Japanese flounder at about 27 kDa (see Figure 3 ).

[0039] Comparative Example 1: Exosomes from fish blood

[0040] Extraction and separation: Steps 1 and 2 are the same as steps 1 and 2 in Example 1; Step 3, the flounder is anesthetized with the anesthetic MS-222, peripheral blood is extracted along the tail vein with a 10 mL syringe (injection needle mark: 0.8×28 mm), and then placed on ice for use; Step 4 is the same as step 5 in Example 1, and finally fish blood-derived exosomes are isolated.

[0041] Detection: Steps 1 and 2 are the same as steps 1 and 2 of Example 2, and the isolated exosome samples are subjected to transmission electron microscopy observation and nanoflow cytometry detection.

[0042] Transmission electron microscopy results showed that compared with exosomes isolated from peritoneal fluid, exosomes isolated from fish blood showed a typical "cup-and-saucer" structure, but the number was significantly smaller (see Figure 4 ); Nanoflow cytometry results also showed that the number of exosomes isolated decreased from 6707 per microliter to 2174 per microliter, which was too small to meet the needs of subsequent functional experiments (see Figure 5 ).

[0043] Comparative Example 2: Effects of different stimuli on the production of exosomes

[0044] Extraction and separation: Step 1, take about 0.5 kg of healthy flounder, and use a 1 mL syringe (injection needle mark: 0.45×15 mm) to intraperitoneally inject an equal amount (200 μL) of phosphate buffered saline (PBS, formula: NaCl 8 g, KCl 0.2 g, KH2PO40.2 g, Na2HPO4•12H2O 2.9 g) as a control, and induce for 6-24 hours; the subsequent steps are the same as steps 3, 4, and 5 in Example 1, and finally the exosomes produced by PBS as the stimulus source are isolated.

[0045] Detection: The steps are the same as step 3 in Example 2, and the isolated exosome samples are identified by Western blotting.

[0046] The results showed that compared with the exosomes produced by PBS stimulation in the control group (Comparative Example 2), fish peritoneal cells can release more exosomes rich in antigen presenting molecules MHCII positive after stimulation with inactivated Vibrio anguillarum (Example 1), which can meet the needs of subsequent biological function research (see Figure 6 ).

[0047] By comparison, it can be found that the present invention creatively establishes an efficient and high-yield method for isolating marine fish-rich MHCII-positive exosomes with antigen presenting molecules through stimulants suitable for fish and a unique fish peritoneal lavage method, which solves the problem of being unable to obtain a large number of exosomes and conduct biological function research in the past, and achieves good results.

[0048] Effect Example 1: Effect of exosomes rich in antigen presenting molecules MHCII positive on lymphocyte activation

[0049] Step 1: Select a healthy and healthy flounder, draw peripheral blood from the tail vein, and quickly put the syringe into the clean bench, inject it into the filtered anticoagulant (RPMI-1640 100mL, BSA 1g, heparin sodium 0.07g), and incubate at 4℃ and 100× g Centrifuge for 10 minutes under the conditions to remove the red blood cells. After the centrifugation, aspirate the supernatant in the clean bench and place it in an ice box for later use. Take several sterilized 10mL centrifuge tubes, fully wet the tube walls with filtered fetal bovine serum, and use a pipette to aspirate the serum remaining at the bottom. Add 3mL of filtered 1.070 g / mL Percoll separation solution, 3mL of 1.020 g / mL Percoll separation solution, and 3mL of supernatant to the centrifuge tubes. Incubate at 4°C and 840× g Under the conditions, centrifuge for 30 minutes. After the centrifugation, aspirate the white film layer and collect it into a 10mL centrifuge tube. Each centrifuge tube is filled with 4mL, and then add an equal amount of 1640 culture medium and incubate at 4℃ and 680× g The cells were centrifuged for 5 min and washed. Finally, they were resuspended in 1640 culture medium. The collected cells were peripheral blood lymphocytes of flounder. The cell concentration was adjusted to 1x10 7 / mL.

[0050] Step 2: Add 1 μL of CFSE dye per ml of cells, mix thoroughly with a pipette, and incubate in a 37°C incubator.

[0051] 30min, stain the cells. Add 5mL of pre-cooled 1640 culture medium to the centrifuge tube, mix thoroughly with a pipette, and place on an ice box for 5min to terminate the staining process. Incubate the cells at 4℃ and 680× g Centrifuge for 5 min under the same conditions. Pour off the supernatant, resuspend the cells with PBS solution, and centrifuge and resuspend the cells again under the same conditions to thoroughly wash away the excess dye. Pipette 100 μL of the cell suspension into a 0.5 mL centrifuge tube and perform flow cytometry to detect the labeling effect of CFSE dye on the cells. At the same time, count the cells and adjust the cell concentration to 5×10 6 / mL, add dropwise into a 24-well plate, 1mL per well.

[0052] Step 3: Add the exosomes extracted by the above method to lymphocytes and culture them at 20°C in the dark for 3 days, during which the culture status of the cells is carefully observed. After 3 days of culture, the cells are aspirated into a 1.5 mL centrifuge tube.

[0053] Step 4, 4°C, 680×g The lymphocytes were precipitated by centrifugation for 5 min under the same conditions, and the cell pellets were resuspended with 1 mL of mouse anti-CD4-1 (1:1000) and CD4-2 (1:1000) antibodies, respectively, incubated at 37°C for 1 h, and washed three times with sterile PBS. g After centrifugation for 5 min under the same conditions, the cell pellet was resuspended with fluorescein-labeled IgG antibody and incubated at 37°C for 1 h. After washing away the unbound secondary antibody with PBS, the positive signal of the cells was detected using an Accuri C6 flow cytometer (BD Accuri, USA). Non-immune mouse serum was used instead of the primary antibody as a negative control.

[0054] The results showed that after 3 days of co-incubation, leukocytes were able to divide twice (see Figure 7 A); Analysis of CD4-1 by flow cytometry + T and CD4-2 + Compared with the control group (3.50±0.50%), the MHCII-enriched exosome group (7.27±0.31%) significantly promoted the expression of CD4-1 + Increase of T cells ( P <0.05), CD4-2 + The proportion of T cells was 3.27±0.31%, and CD4-2 was enriched in MHCII exosomes. + The proportion of T lymphocytes increased to 10.33±0.42% (see Figure 7 B). The above experiments prove that the method for separating MHCII-positive exosomes rich in antigen presenting molecules provided in the present invention can effectively promote CD4 + T lymphocyte activation.

[0055] Effect Example 2: The role of positive exosomes rich in antigen presenting molecules MHCII in promoting vaccine immune efficacy

[0056] Step 1: After temporary rearing, the flounder with good vitality was randomly divided into 4 groups. After the flounder was anesthetized, it was immunized by intraperitoneal injection. Each flounder was intraperitoneally injected with inactivated Vibrio anguillarum, MHCII-rich exosomes, and a mixture of inactivated Vibrio anguillarum and MHCII-rich exosomes. An equal amount of PBS was injected as a control. After injection, pay attention to observe whether there is fluid outflow from the injection site. Flow cytometry was used to examine the CD4 at different time points after immunization. + T and IgM + Changes in the proportion of B cells.

[0057] Step 2: To detect the role of MHCII-rich exosomes in promoting vaccine immunity on CD4 +In order to investigate the effect of changes in the proportion of T lymphocytes, lymphocytes in spleen and head kidney were separated by discontinuous Percoll gradient density (1.020-1.070 g / mL) on days 1, 3, 5, 7, 14 and 28 after immunization. The specific separation steps are shown in Example 1. The cell concentration was adjusted to 1×10 7 / mL, and incubated with mouse anti-CD4-1 (1:1000) and CD4-2 (1:1000) antibodies (primary antibodies). After incubation, cells were incubated at 680× g The primary antibody was removed by centrifugation for 5 min, and the cells were washed three times with PBS. Then, the cells were resuspended with 500 μL of Dylight488 (Abbkine) mouse IgG antibody (secondary antibody) and incubated at 37°C for 1 h. The cells were centrifuged to remove the secondary antibody, and then washed three times with PBS for flow cytometry. FACSCalibur (BD Biosciences) flow cytometer was used to monitor the CD4-1 + T and CD4-2 + Positive signal of T lymphocytes. Mouse negative serum was used as negative treatment for flow cytometry.

[0058] Step 3: To detect the role of MHCII-rich exosomes in promoting vaccine immunity to IgM + In order to investigate the effect of changes in the proportion of B lymphocytes, lymphocytes in peripheral blood and spleen were separated by discontinuous Percoll gradient density (1.020-1.070 g / mL) at 1, 2, 3, and 4 weeks after immunization. The cells were adjusted to 1×10 7 / mL, the cells were centrifuged and incubated with 500 μL of anti-IgM monoclonal antibody (diluted 1:1000). Then, they were stained with Dylight 488 (Abbkine) mouse IgG antibody at 37°C for 1 h. FACSCalibur (BD Biosciences) flow cytometer was used to monitor the expression of IgM in peripheral blood and spleen of different treatment groups. + B lymphocyte positive signal. Mouse negative serum was used as negative treatment for flow cytometry.

[0059] The results showed that compared with the control group, the CD4-1 + T and CD4-2 + T lymphocytes showed a trend of increasing first and then decreasing. On the third day after immunization, CD4-1 + T and CD4-2 + The proportion of T lymphocytes began to rise and reached a peak on the 7th day after immunization. + T and CD4-2+ The proportion of T lymphocytes began to decrease. + The proportion of T lymphocytes at the peak of proliferation was 5.03±0.67% and 7.67±0.42% in the inactivated Vibrio anguillarum group, 12.20±1.02% and 10.40±0.80% in the exosome group alone, and 10.9±0.20% and 13±1.73% in the exosome and inactivated Vibrio anguillarum group; CD4-2 + The proportion of T lymphocytes at the peak of proliferation was 4.33±0.25% and 7.67±0.35% in the inactivated Vibrio anguillarum group, 14.00±0.51% and 15.9±0.27% in the exosomes alone, and 19.00±1.00% and 18.33±1.53% in the exosomes and inactivated Vibrio anguillarum group. + T and CD4-2 + The peak proportion of T lymphocytes was higher than that of the control group and the difference was significant (see Figure 8 ).

[0060] Flow cytometric analysis results showed that the IgM in peripheral blood and spleen of flounder in three experimental groups (killed Vibrio anguillarum, exosomes, and a mixture of exosomes and killed Vibrio anguillarum) + The proportion of B lymphocytes showed a trend of first increasing and then decreasing. Immunity reached its peak in the second week. At the peak, sIgM in peripheral blood and spleen + The proportion of B lymphocytes in the inactivated Vibrio anguillarum group was 50.8±1.40% and 52.2±2.1%, in the exosome group alone was 55.3±5.56% and 51.6±1.22%, and in the exosome and inactivated Vibrio anguillarum mixed group was 52.4±2.51% and 58.07±2.70%, and then the proportion of positive lymphocytes began to decrease. In addition, the sIgM in the peripheral blood and spleen of the experimental group of flounder + The peak value of B lymphocytes was higher than that of the control group and the difference was significant (see Figure 8 ).

[0061] The above results prove that the method for preparing MHCII-positive exosomes rich in antigen presenting molecules provided in the present invention can separate and obtain MHCII-positive exosomes, which play a significant role in promoting the immune efficacy of vaccines.

Claims

1. A method for preparing fish antigen presenting molecule MHCII positive exosomes, characterized in that: The following steps are involved: Step 1: inactivate Vibrio anguillarum by shaking it to the logarithmic growth phase; Step 2: injecting the inactivated Vibrio anguillarum solution into the fish intraperitoneally for 6-24 hours; the fish is flounder; Step 3: After the fish is anesthetized, peripheral blood is drawn along the tail vein with a syringe; after the peripheral blood is removed, serum-free L15 culture medium is injected into the abdominal cavity of the fish with a syringe; Step 4: After massaging the fish's abdomen, use a syringe to draw out the peritoneal fluid along the upper edge of the abdominal cavity; Step 5: Separation of exosomes by continuous gradient centrifugation: At 4°C, the extracted peritoneal fluid was centrifuged at 500× g Centrifuge for 10 min, 4000× g Centrifuge for 30 min to remove cells and cell debris to obtain the supernatant; Cell supernatant 10000× g Centrifuge for 30 min to remove the large vesicles in the fish peritoneal cavity, and the cell supernatant is further heated at 115000× g Centrifuge for 90 min to obtain a precipitate; resuspend the precipitate in phosphate buffer and centrifuge at 115,000 × g After centrifugation for 90 min, the supernatant containing liposomes was removed, and the precipitate was resuspended in phosphate buffer to separate the positive exosomes rich in fish antigen presenting molecule MHCII.

2. The method for preparing exosomes according to claim 1, characterized in that: The inactivation method of Vibrio anguillarum is to use formaldehyde solution at 37° C. and 180 rpm to inactivate it.

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