Preparation method of artificial exosome of crustacean blood cells

Abstract

The application belongs to the technical field of exosomes, and discloses a preparation method of artificial exosomes of blood cells of a crustacean Scylla paramamosain, which mainly comprises the following steps: 1) collecting blood cells of the Scylla paramamosain by centrifugation; 2) collecting cell membranes and cytoplasmic matrix respectively after blood cells are broken and centrifuged; and 3) obtaining artificial exosomes under certain reaction conditions. The exosomes obtained by the production method can be used as a carrier, and by carrying RNA or proteins, can be widely used in immunological research and disease prevention and treatment of the crustacean.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of exosomes and particularly relates to a preparation method of artificial exosomes of blood cells of crustaceans. BACKGROUND

[0002] In addition to gene therapy, small molecule drugs and macromolecular drugs cannot be separated from the dependence on delivery technology. From the delivery of small molecule chemical agents by in vitro microneedle injection to the delivery of macromolecular drugs by encapsulating them with liposomes (LNP), delivery technology has become an indispensable part of the medical industry. The delivery effect depends on both the delivered substance and the efficiency and safety of the carrier. At present, most delivery technologies use viruses as carriers. Traditional carriers include viral vectors and non-viral vectors, but viral vectors may insert mutations, and non-viral vectors are difficult to achieve low toxicity and high transfection efficiency at the same time. Therefore, it is necessary to develop new delivery carriers.

[0003] Exosomes are extracellular membrane vesicles with a diameter of 30-150 nm and play an important role in intercellular information exchange. Exosomes are similar to liposomes in non-viral carriers, but are more complex in structure and function. Since exosomes are similar in size and function to synthetic nanoparticles, as natural endogenous transport carriers, they have the advantages of low toxicity, no immunogenicity, and good permeability, and are now considered the most promising drug delivery carrier, suitable for delivering various chemicals, proteins, nucleic acids, and gene therapy agents.

[0004] A key problem of exosomes as delivery carriers is how to obtain exosomes with high yield and high purity. This is mainly due to the relatively low number of exosomes released by cells, the difficulty in purifying exosomes, and the limited ability to load various cargoes. Exosomes have good application prospects in precision medicine and targeted therapy of tumors, but there is less research on aquatic animals. Research on artificial exosomes in crustaceans is of great significance for building a crustacean research platform and using exosomes for disease prevention and control. SUMMARY

[0005] The purpose of the present application is to solve the problems of difficult exosome acquisition, low yield, and limited ability to load various cargoes, and to provide a preparation method of artificial exosome of blood cells of the mud crab Scylla paramamosain. This method uses natural cell membranes and cytoplasm to produce exosomes that can carry active substances. These exosomes are non-toxic and can be efficiently taken up by recipient cells, bringing active substances into recipient cells and exerting therapeutic effects.

[0006] To solve the above technical problems, the present application adopts the following technical solutions:

[0007] A preparation method of artificial exosomes of blood cells of crustaceans, comprising the following steps:

[0008] A, collecting blood cells;

[0009] B, separating the cell membrane and cytoplasmic matrix of the blood cells;

[0010] C, adding the cell membrane and the cytoplasmic matrix to a reaction system to obtain artificial exosomes by reaction.

[0011] Preferably, in step B, the reaction system comprises a regeneration system and an incubation buffer.

[0012] Preferably, the regeneration system is an ATP regeneration system, comprising ATP, GDP-mannose, creatine phosphate, creatine phosphokinase, HEPES-pH 7.2, sorbitol, potassium acetate, magnesium acetate; and the incubation buffer comprises KCl, CaCl2, HEPES-NaOH-pH 7.4, MgOAc, DTT.

[0013] Preferably, the ATP regeneration system comprises 10 mM ATP, 500 mM GDP-mannose, 400 mM creatine phosphate, 40 mM creatine phosphokinase, 20 mM HEPES-pH 7.2, 250 mM sorbitol, 150 mM potassium acetate, 5 mM magnesium acetate; and the incubation buffer comprises 80 mM KCl, 20 mM CaCl2, 12.5 mM HEPES-NaOH-pH 7.4, 1.5 mM MgOAc, 1 mM DTT.

[0014] It is extremely difficult to synthesize artificial exosomes of crustacean blood cells, and the synthesis cannot be achieved if the ATP concentration or the pH of the buffer is not appropriate. Through long-term research and continuous adjustment of the proportion, the present application finally determines the appropriate ATP regeneration system and incubation buffer, and successfully synthesizes the artificial exosomes of crustacean blood cells.

[0015] Preferably, in step B, a carrier is further added to the reaction system, and the carrier comprises one or more of nucleic acids and proteins.

[0016] Preferably, the carrier comprises one or more of 14-3-3 protein, MyD88 protein, CYP-siRNA, and STEAP4-siRNA.

[0017] The loading experiments of 14-3-3 protein and MyD88 protein show that the artificial exosomes synthesized by the present application can well load proteins and keep them active and play the corresponding roles. The loading experiments of CYP-siRNA and STEAP4-siRNA show that the artificial exosomes synthesized by the present application can also well load nucleic acids and keep them active and play the corresponding roles.

[0018] Preferably, the method comprises the following steps:

[0019] A, collecting the blood cells:

[0020] A1, extracting blood from a crustacean, mixing with an anticoagulant, and centrifuging to collect the precipitate;

[0021] A2, resuspending the precipitate obtained in step A1 with an anticoagulant, centrifuging to collect the precipitate, and obtaining the blood cells;

[0022] B, separating the cell membrane and the cytoplasmic matrix:

[0023] B1, resuspending the blood cells with a homogenizing solution and breaking the blood cells by ultrasonic oscillation on ice to obtain a broken cell solution;

[0024] B2, centrifuging the broken cell solution and collecting the supernatant;

[0025] B3, centrifuging the supernatant obtained in step B2, collecting the supernatant and the precipitate;

[0026] B4, centrifuging the supernatant obtained in step B3, collecting the supernatant, and obtaining a solution of the cytoplasmic matrix;

[0027] B5, resuspending the precipitate obtained in step B3 with the homogenizing solution and a LiCl solution, centrifuging, and resuspending with the homogenizing solution to obtain a solution of the cell membrane

[0028] C, adding the solution of the cytoplasmic matrix and the solution of the cell membrane to an ATP regeneration system and an incubation buffer, and then adding a carrier to obtain the artificial exosome by reaction, and cryopreserving.

[0029] Preferably, in step A1, the crustacean is Scylla paramamosain, the volume ratio of blood to anticoagulant after blood extraction is 1:1, and the centrifugation operation is 1000xg centrifugation for 10 min; in step A2, the volume ratio of anticoagulant to the obtained precipitate in step A1 for resuspension is 1000:1, and the centrifugation operation is 1000xg centrifugation for 10 min; in step B1, the volume ratio of the homogenate to the blood cells is 10:1; in step B1, the ultrasonic operation is 3s on and 2s off for 3 min; in step B2, the centrifugation operation is 1500xg centrifugation for 10 min; in step B3, the centrifugation operation is 20000xg centrifugation for 30 min; in step B4, the centrifugation operation is 60000xg centrifugation for 30 min; in step B5, the volume ratio of the homogenate, the LiCl solution and the precipitate before resuspension is 2:1:1, and the volume ratio of the homogenate and the precipitate after resuspension is 5:1; in step B5, the centrifugation operation is 20000xg centrifugation for 30 min; in step C, the volume ratio of the solution of the cytoplasmic matrix, the solution of the cell membrane, the ATP regeneration system, the incubation buffer and the carrier is 17:10:40:8; in step C, the temperature for cryopreservation is -80℃.

[0030] If the amount of ATP is too small, the artificial exosome cannot be successfully synthesized.

[0031] Preferably, the homogenate comprises 250mM sorbitol, 137mM NaCl and 10mM PMSF, and is dissolved with Tris-HCl-pH7.4.

[0032] A method for preparing the artificial exosome of the crustacean blood cells as described above obtains the artificial exosome.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] The exosome carrying the bioactive substance can be easily taken by the recipient cells to play the role of the bioactive substance, and the exosome itself has no significant immune rejection. The exosome carrying the bioactive substance can be widely used as a new type of delivery tool for immunological research and treatment of various diseases. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 A method flowchart for the present application;

[0036] Figure 2 The artificial exosome can be absorbed by the blood cells of Scylla paramamosain; the artificial exosome membrane is dyed in vitro, and then injected into the body of Scylla paramamosain, and 3h later, the blood cells of Scylla paramamosain are extracted and observed by a confocal microscope to observe whether there are dyed exosomes in the blood cells of Scylla paramamosain;

[0037] Figure 3 A is the image of artificial exosome carrying 14-3-3 protein (high-abundance protein in blue crab blood exosome, Sun et al., Journal of Immunology. 2022, 209: 710-722.) under transmission electron microscope; B is the image of artificial exosome carrying MyD88 protein under transmission electron microscope; C is the image of artificial exosome carrying CYP-siRNA protein under transmission electron microscope; D is the image of artificial exosome carrying STEAP4-siRNA under transmission electron microscope;

[0038] Figure 4 A is the image of artificial exosome carrying 14-3-3 protein for particle size detection; B is the image of artificial exosome carrying MyD88 protein for particle size detection; C is the image of artificial exosome carrying CYP-siRNA for particle size detection; D is the image of artificial exosome carrying STEAP4-siRNA for particle size detection;

[0039] Figure 5 A is the detection of 14-3-3 protein expression content in blue crab blood cells after artificial exosome carrying 14-3-3 protein is injected into blue crab for 6h; B is the detection of MyD88 protein and MyD88 related TLR protein expression content in blue crab blood cells after artificial exosome carrying MyD88 protein is injected into blue crab for 6h; C is the detection of transcription level changes of anti-lipopolysaccharide factors ALF1, ALF4, ALF5 in blue crab blood cells after artificial exosome carrying 14-3-3 protein is injected into blue crab for 6h;

[0040] Figure 6 A is the detection of CYP transcription level changes in blue crab blood cells after artificial exosome carrying CYP-siRNA is injected into blue crab for 6h; B is the detection of CYP translation level changes in blue crab blood cells after artificial exosome carrying CYP-siRNA is injected into blue crab for 6h. C is the detection of STEAP4 transcription level changes in blue crab blood cells after artificial exosome carrying STEAP4-siRNA is injected into blue crab for 6h; D is the detection of STEAP4 translation level changes in blue crab blood cells after artificial exosome carrying STEAP4-siRNA is injected into blue crab for 6h. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below with reference to the drawings, so that those skilled in the art can better understand the present application and implement it. The embodiments are not intended to limit the present application. Any modification or replacement of the method, step or condition of the present application without departing from the spirit and essence of the present application shall fall within the scope of the present application. If not specifically indicated, the technical means used in the embodiments are conventional means known to those skilled in the art.

[0042] It should be noted that all components used in the following embodiments can be purchased or self-made.

[0043] The artificial exosome preparation method provided in the embodiments of the present application is as shown in the figure, and the method is specifically realized by the following steps: Figure 1

[0044] S1. Centrifugal collection of blood cells of Scylla paramamosain;

[0045] S2. Centrifugal collection of cell membrane and cytoplasmic matrix after cell breakage;

[0046] S3. Obtaining artificial exosomes under certain reaction conditions;

[0047] S4. In vitro staining of the membrane of the artificial exosomes, injection of the artificial exosomes after in vitro staining into the body of Scylla paramamosain, extraction of blood cells of Scylla paramamosain after 3h and observation of the blood cells of Scylla paramamosain by confocal microscopy.

[0048] The results are shown in Figure 2 The results show that the exosomes synthesized in vitro can be absorbed by the blood cells of Scylla paramamosain.

[0049] Embodiment 1

[0050] S1. Cell collection: blood was extracted from the appendage of Scylla paramamosain with a 5mL syringe, 1mL blood was mixed with 1mL anticoagulant, 1000g centrifugal for 10min, the obtained precipitate was resuspended with anticoagulant, and centrifugal for 10min at 1000g again, and the cell precipitate was collected;

[0051] S2. Cell breakage: the above cell precipitate was resuspended with 10 times the volume of homogenate (250mM sorbitol, 137mM NaCl, 10mM PMSF, dissolved with Tris-HCl-pH 7.4) and broken on ice at 5% power for 3min (ultrasonic opening for 3s, closing for 2s);

[0052] S3. Low-speed centrifugation: the solution after breakage in S2 was centrifuged at 1500xg for 10min;

[0053] ​S4. High-speed centrifugation: the supernatant after centrifugation of S3 was centrifuged again at 20000xg for 30min;

[0054] S5. Cytoplasmic solution collection: the supernatant after centrifugation of S4 was centrifuged at 60000xg for 30min and the supernatant after centrifugation was collected;

[0055] S6. Cell membrane solution collection: the precipitate after centrifugation of S4 was resuspended with twice volume of homogenate and one volume of LiCl solution, centrifuged at 20000xg for 30min, and resuspended with 5 times volume of homogenate;

[0056] S7. In vitro reaction: the following reaction system was prepared: 17uL cytoplasmic solution obtained in S5, 10uL cell membrane solution obtained in S6, 4uL ATP regeneration system (10mM ATP, 500mM GDP-mannose, 400mM phosphocreatine, 40mM phosphocreatine kinase, 20mM HEPEs-pH 7.2, 250mM sorbitol, 150mM potassium acetate, 5mM magnesium acetate), 8uL incubation buffer (80mM KCl, 20mM CaCl2, 12.5mM HEPES-NaOH, pH 7.4, 1.5mM, MgOAc, 1mM DTT), 1uL solution of in vitro expressed and purified 14-3-3 protein of Scylla paramamosain (1uL homogenate was added to the control group), and reaction was carried out at 30℃ for 20min;

[0057] S8. Ultracentrifugation was used to collect exosomes carrying 14-3-3 protein. Centrifugation was carried out at 20000xg for 30min, the precipitate was collected and resuspended with 50uL PBS to obtain the artificial exosomes;

[0058] S9. The exosomes obtained above were observed by transmission electron microscopy;

[0059] Results are shown in Figure 3 A. The results showed that the exosomes carrying 14-3-3 protein had a double-membrane structure, and their appearance was similar to that of naturally extracted exosomes.

[0060] S10. The exosomes obtained above were subjected to particle size detection;

[0061] Results are shown in Figure 4 A. The results showed that the diameter of the exosomes carrying 14-3-3 protein was 50-200nm, which was similar to that of naturally extracted exosomes.

[0062] S11. The exosomes obtained above were injected into Scylla paramamosain;

[0063] S12. The blood cells of the Scylla paramamosain above were collected after 6h, and blood cell lysate was prepared;

[0064] S13. Detect the content of 14-3-3 protein in the above blood cell lysate using Western blot;

[0065] Results are shown in Figure 5 A. The results show that the exosomes carrying 14-3-3 protein can increase the content of 14-3-3 protein in the blood cells of Scylla paramamosain.

[0066] S15. Detect the downstream regulation signal pathway (expression of antibacterial peptide) of 14-3-3 protein in the blood cells of Scylla paramamosain; results are shown in Figure 5 C. The results show that the exosomes carrying 14-3-3 protein can increase the expression content of 14-3-3 downstream regulated antibacterial peptides ALF1, ALF4 and ALF5 in the blood cells of Scylla paramamosain.

[0067] Example 2

[0068] S1. Cell collection: 5 mL syringe was used to extract blood from the appendage of Scylla paramamosain, 1 mL blood was mixed with 1 mL anticoagulant, 1000g centrifugation for 10 min, the anticoagulant was used to resuspend the obtained precipitate, and 1000g centrifugation for 10 min again, and the cell precipitate was collected;

[0069] S2. Cell disruption: the above cell precipitate was resuspended with 10 times volume of homogenate (250mM sorbitol, 137mM NaCl, 10mM PMSF, dissolved with Tris-HCl-pH 7.4) and disrupted on ice at 5% power for 3 min (ultrasound on for 3s, off for 2s);

[0070] S3. Low speed centrifugation: the solution after disruption in S2 was centrifuged at 1500xg for 10 min;

[0071] S4. High speed centrifugation: the supernatant after centrifugation in S3 was centrifuged at 20000xg for 30 min again;

[0072] S5. Cytoplasmic solution collection: the supernatant after centrifugation in S4 was centrifuged at 60000xg for 30 min and the supernatant after centrifugation was collected;

[0073] S6. Cell membrane solution collection: the precipitate after centrifugation in S4 was resuspended with twice volume of homogenate and one volume of LiCl solution, centrifuged at 20000xg for 30 min, and resuspended with 5 times volume of homogenate;

[0074] S7. In vitro reaction: prepare the following reaction system: 17uL of cytoplasmic solution obtained in S5, 10uL of cell membrane solution obtained in S6, 4uL of ATP regeneration system (10mM ATP, 500mM GDP-mannose, 400mM phosphocreatine, 40mM creatine phosphokinase, 20mM HEPEs-pH 7.2, 250mM sorbitol, 150mM potassium acetate, 5mM magnesium acetate); 8uL of incubation buffer (80mM KCl, 20mM CaCl2, 12.5mM HEPES-NaOH, pH 7.4, 1.5mM, MgOAc, 1mM DTT); add 1uL of in vitro expressed and purified MyD88 protein solution of Scylla paramamosain (add 1uL of homogenate to the control group), and react at 30°C for 20min;

[0075] S8. Collect the exosomes carrying MyD88 protein by ultracentrifugation. Centrifuge at 20000xg for 30min, collect the precipitate and resuspend with 50uL of PBS to obtain the artificial exosomes;

[0076] S9. Observe the exosomes obtained above by transmission electron microscopy;

[0077] Results are shown in Figure 3 B. The results show that the exosomes carrying MyD88 protein have a double-membrane structure, and their appearance is similar to that of naturally extracted exosomes.

[0078] S10. Detect the particle size of the exosomes obtained above;

[0079] Results are shown in Figure 4 B. The results show that the diameter of the exosomes carrying MyD88 protein is 50-200nm, which is similar to that of naturally extracted exosomes.

[0080] S11. Inject the exosomes obtained above into Scylla paramamosain;

[0081] S12. Collect the blood cells of the Scylla paramamosain 6h later, and prepare blood cell lysate;

[0082] S13. Detect the MyD88 protein content in the blood cell lysate by Western blot;

[0083] Results are shown in Figure 5 B. The results show that the exosomes carrying MyD88 protein can increase the 14-3-3 protein content in the blood cells of Scylla paramamosain.

[0084] S15. Detect the MyD88 protein downstream regulation signal pathway (expression of antibacterial peptide) in the blood cells of Scylla paramamosain; results are shown in Figure 5B. The results show that the MyD88 protein-carrying exosomes can increase the expression content of TLR, the MyD88 interacting protein in the blood cells of Scylla paramamosain.

[0085] Example 3

[0086] S1. Cell collection: 5 mL syringe was used to extract blood from the appendage of Scylla paramamosain, 1 mL blood was mixed with 1 mL anticoagulant, centrifuged at 1000xg for 10 min, the anticoagulant was used to resuspend the obtained precipitate, and centrifuged at 1000xg for 10 min again, and the cell precipitate was collected;

[0087] S2. Cell disruption: the above cell precipitate was resuspended with 10 times volume of homogenate (250 mM sorbitol, 137 mM NaCl, 10 mM PMSF, dissolved with Tris-HCl-pH 7.4) and disrupted on ice at 5% power for 3 min (ultrasonic open for 3 s, close for 2 s);

[0088] S3. Low-speed centrifugation: the solution after disruption in S2 was centrifuged at 1500xg for 10 min;

[0089] S4. High-speed centrifugation: the supernatant after centrifugation in S3 was centrifuged at 20000xg for 30 min again;

[0090] S5. Cytoplasmic solution collection: the supernatant after centrifugation in S4 was centrifuged at 60000xg for 30 min and the supernatant after centrifugation was collected;

[0091] S6. Cell membrane solution collection: the precipitate after centrifugation in S4 was resuspended with two times volume of homogenate and one times volume of LiCl solution, centrifuged at 20000xg for 30 min, and resuspended with 5 times volume of homogenate;

[0092] S7. In vitro reaction: the following reaction system was prepared: 17 uL cytoplasmic solution obtained in S5, 10 uL cell membrane solution obtained in S6, 4 uL 10x ATP regeneration system (10 mM ATP, 500 mM GDP-mannose, 400 mM phosphocreatine, 40 mM creatine phosphokinase, 20 mM HEPEs-pH 7.2, 250 mM sorbitol, 150 mM potassium acetate, 5 mM magnesium acetate), 8 uL incubation buffer (80 mM KCl, 20 mM CaCl2, 12.5 mM HEPES-NaOH, pH 7.4, 1.5 mM MgOAc, 1 mM DTT), 1 uL in vitro synthesized Scylla paramamosain CYP-siRNA (1 uL homogenate was added to the control group), and reacted at 30°C for 20 min;

[0093] S8. Collect the exosomes carrying CYP-siRNA by ultracentrifugation. Centrifuge at 20000xg for 30min, collect the precipitate and resuspend with 50uL PBS, and obtain the artificial exosomes;

[0094] S9. Observe the exosomes obtained above by transmission electron microscopy sample preparation;

[0095] Results are shown in Figure 3 C. The results show that the exosomes carrying CYP-siRNA have a double-membrane structure, and their appearance is similar to that of naturally extracted exosomes.

[0096] S10. Detect the particle size of the exosomes obtained above;

[0097] Results are shown in Figure 4 C. The results show that the diameter of the exosomes carrying CYP-siRNA is 50-200nm, which is similar to that of naturally extracted exosomes.

[0098] S11. Inject the exosomes obtained above into the Scylla paramamosain;

[0099] S12. Collect the blood cells of the Scylla paramamosain 6h later, and extract RNA;

[0100] S13. Detect the mRNA content of CYP in the blood cells above by qPCR;

[0101] Results are shown in Figure 6 A. The results show that the exosomes carrying CYP-siRNA can reduce the mRNA content of CYP in the blood cells of Scylla paramamosain.

[0102] S14. Detect the protein content of CYP in the above S12 by Western blot;

[0103] Results are shown in Figure 6 B. The results show that the exosomes carrying CYP-siRNA can reduce the protein content of CYP in the blood cells of Scylla paramamosain.

[0104] Example 4

[0105] S1. Cell collection: draw blood from the appendage of Scylla paramamosain with a 5mL syringe, mix 1mL blood with 1mL anticoagulant, centrifuge at 1000xg for 10min, resuspend the precipitate with anticoagulant, centrifuge again at 1000xg for 10min, and collect the cell precipitate;

[0106] S2. Cell disruption: resuspend the cell precipitate above with 10 times the volume of homogenate (250mM sorbitol, 137mM NaCl, 10mM PMSF, dissolved with Tris-HCl-pH 7.4), and disrupt on ice at 5% power for 3min (ultrasound on for 3s, off for 2s);

[0107] S3. Low speed centrifugation: centrifuge the broken solution in S2 at 1500xg for 10 min;

[0108] S4. High speed centrifugation: centrifuge the supernatant after S3 at 20000xg for 30 min;

[0109] S5. Cytoplasmic solution collection: centrifuge the supernatant after S4 at 60000xg for 30 min and collect the supernatant after centrifugation;

[0110] S6. Cell membrane solution collection: resuspend the precipitate after S4 with two volumes of homogenate and one volume of LiCl solution, centrifuge at 20000xg for 30 min, and resuspend with 5 volumes of homogenate;

[0111] S7. In vitro reaction: prepare the following reaction system: 17uL of cytoplasmic solution obtained in S5, 10uL of cell membrane solution obtained in S6, 4uL of 10x ATP regeneration system (10mM ATP, 500mM GDP-mannose, 400mM phosphocreatine, 40mM phosphocreatine kinase, 20mM HEPEs-pH 7.2, 250mM sorbitol, 150mM potassium acetate, 5mM magnesium acetate), 8uL of incubation buffer (80mM KCl, 20mM CaCl2, 12.5mM HEPES-NaOH, pH 7.4, 1.5mM, MgOAc, 1mM DTT), add luL of in vitro synthesized STEAP4-siRNA (add 1uL of homogenate to the control group), react at 30°C for 20 min;

[0112] S8. Collect the exosomes carrying STEAP4-siRNA by ultracentrifugation. Centrifuge at 20000xg for 30 min, collect the precipitate and resuspend with 50uL of PBS to obtain the artificial exosomes;

[0113] S9. Observe the exosomes obtained above by transmission electron microscopy sample preparation;

[0114] Results are shown in Figure 3 D. The results show that the exosomes carrying STEAP4-siRNA have a double-membrane structure, and their appearance is similar to that of naturally extracted exosomes.

[0115] S10. Detect the particle size of the exosomes obtained above;

[0116] Results are shown in Figure 4 D. The results show that the diameter of the exosomes carrying STEAP4-siRNA is 50-200nm, which is similar to that of naturally extracted exosomes.

[0117] S11. Injecting the above obtained exosomes into the Scylla paramamosain;

[0118] S12. Collecting the blood cells of the Scylla paramamosain after 6h, and extracting RNA;

[0119] S13. Detecting the mRNA content of STEAP4 in the blood cells by qPCR;

[0120] The results are shown in Figure 6 C. The results show that the exosomes carrying STEAP4-siRNA can reduce the mRNA content of STEAP4 in the blood cells of the Scylla paramamosain.

[0121] S14. Detecting the protein content of STEAP4 in the blood cells of the Scylla paramamosain by Western blot;

[0122] The results are shown in Figure 6 D. The results show that the exosomes carrying STEAP4-siRNA can reduce the protein content of STEAP4 in the blood cells of the Scylla paramamosain.

[0123] In summary, the exosomes carrying bioactive substances can be effectively produced by the production method of the present application; the exosomes can be easily taken up by the recipient cells to play the role of bioactive substances, and the exosomes themselves have no significant immune rejection. The exosomes carrying bioactive substances can be used as a new type of delivery tool for immunological research and treatment of various diseases.

[0124] The above only discloses the preferred embodiments of the present application, and of course cannot limit the scope of the rights of the present application, so the equivalent changes made according to the claims of the present application still fall within the scope of the present application.

Claims

1. A method for preparing artificial exosomes from crustacean blood cells, characterized in that, Includes the following steps: A. Collecting blood cells; B. Separate the cell membrane and cytoplasm of the blood cells; C. The cell membrane and the cytoplasm are added to the reaction system to obtain artificial exosomes; In step B, the reaction system includes a regeneration system and an incubation buffer; the regeneration system is an ATP regeneration system; the ATP regeneration system includes 10 mM ATP, 500 mM GDP-mannose, 400 mM creatine phosphate, 40 mM creatine phosphokinase, 20 mM HEPEs-pH 7.2, 250 mM sorbitol, 150 mM potassium acetate, and 5 mM magnesium acetate; the incubation buffer includes 80 mM KCl, 20 mM CaCl2, 12.5 mM HEPES-NaOH-pH 7.4, 1.5 mM MgOAc, and 1 mM DTT; step C further includes adding a carrier to the reaction system, the carrier including one or more of nucleic acids and proteins.

2. The method for preparing artificial exosomes from crustacean blood cells as described in claim 1, characterized in that, The vector includes one or more of the following: 14-3-3 protein, MyD88 protein, CYP-siRNA, and STEAP4-siRNA.

3. The method for preparing artificial exosomes from crustacean blood cells as described in claim 1, characterized in that, Includes the following steps: A. Collect the blood cells: A1. Blood is drawn from crustaceans, mixed with an anticoagulant, and the precipitate is collected by centrifugation. A2. Resuspend the precipitate obtained in step A1 with an anticoagulant, centrifuge to collect the precipitate, and obtain the blood cells; B. Separate the cell membrane and the cytoplasm: B1. Resuspend the blood cells in a homogenizing solution and break them up by ultrasonic vibration on ice to obtain a broken cell solution. B2. Centrifuge the broken cell solution and collect the supernatant; B3. Centrifuge the supernatant obtained in step B2 and collect the supernatant and precipitate; B4. Centrifuge the supernatant obtained in step B3, collect the supernatant, and obtain the solution of the cytoplasmic matrix; B5. Resuspend the precipitate obtained in step B3 with the homogenizing solution and LiCl solution, centrifuge, and resuspend with the homogenizing solution to obtain the cell membrane solution; C. Add the cytoplasmic matrix solution and the cell membrane solution to the ATP regeneration system and incubation buffer, then add the carrier, react to obtain the artificial exosomes, and freeze them.

4. The method for preparing artificial exosomes from crustacean blood cells as described in claim 3, characterized in that, In step A1, the crustacean is a mud crab (Scylla serrata), and the volume ratio of blood drawn to anticoagulant is 1:

1. The centrifugation operation is performed at 1000 × 10⁻⁶. g Centrifuge for 10 min; in step A2, resuspend the precipitate obtained in step A1 with an anticoagulant at a volume ratio of 1000:1, and centrifuge at 1000× g Centrifuge for 10 min; in step B1, the volume ratio of the homogenized solution to the blood cells is 10:1; in step B1, the sonication operation is to turn on for 3 seconds, turn off for 2 seconds, and continue for 3 min; in step B2, the centrifugation operation is 1500 × 10⁻⁶. g Centrifuge for 10 minutes; In step B3, the centrifugation operation is 20000 × g Centrifuge for 30 minutes; in step B4, the centrifugation operation is 60000 × g Centrifuge for 30 min; in step B5, the volume ratio of the resuspended homogenate, the LiCl solution, and the precipitate before centrifugation is 2:1:1, and the volume ratio of the resuspended homogenate to the precipitate after centrifugation is 5:1; in step B5, the centrifugation operation is 20000 × g Centrifuge for 30 min; in step C, the volume ratio of the cytoplasmic matrix solution, the cell membrane solution, the ATP regeneration system, the incubation buffer, and the carrier is 17:10:5:8; in step C, the cryopreservation temperature is -80℃.

5. The method for preparing artificial exosomes from crustacean blood cells as described in claim 3, characterized in that, The homogenizing solution comprises 250 mM sorbitol, 137 mM NaCl, and 10 mM PMSF, dissolved in Tris-HCl at pH 7.4.

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