Sa-tim4 protein, preparation method thereof and application of sa-tim4 protein in isolating exosomes

Abstract

This invention belongs to the field of recombinant protein and its application technology, specifically relating to an SA-Tim4 protein, its preparation method, and its application in exosome isolation. Its amino acid sequence is shown in SEQ ID NO.1. This invention provides an SA-Tim4 protein, its preparation method, and its application in exosome isolation, enabling efficient preparation of SA-Tim4, overcoming the high production cost problem of existing separation technologies, and breaking through the limitations of traditional exosome isolation technologies, such as time consumption, high requirements for equipment and reaction conditions, and difficulty in large-scale separation.

Description

Technical Field

[0001] This invention belongs to the field of recombinant protein and its application technology, specifically relating to an SA-Tim4 protein, its preparation method, and its application in the isolation of exosomes. Background Technology

[0002] Exosomes are extracellular vesicles formed by cells through autocrine or paracrine processes. They are approximately 30–150 nm in diameter and contain abundant DNA, RNA, and proteins that participate in cellular signal transduction and regulation of the immune system. They can be used for disease diagnosis and treatment, drug delivery, and tissue repair. Therefore, exosomes have broad research prospects in the fields of food, medicine, pharmaceuticals, and biology.

[0003] Exosome isolation technology is crucial for the widespread application of exosomes, and efficient, portable, high-purity, and large-scale preparation techniques have become research hotspots. Existing exosome isolation technologies mainly focus on separation based on density differences, solubility, and fluid properties, which suffer from low purity, high equipment requirements, and long processing times. In contrast, affinity separation techniques based on immunoreactions can specifically separate exosomes using the antigen-antibody specific binding immune reaction. Chinese patents CN113046303A ("A Rapid Extraction Kit for Exosome Isolation") and CN111849903A ("A Kit for Separating Exosomes from Cell Supernatant and Its Usage Method") both mention using recombinant TIMD4 protein, recombinant TIMD3 protein, or Annexin V protein as components for recognizing and binding exosomes. However, the recombinant TIMD4, TIMD3, or Annexin V proteins lack relative specificity, have relatively fixed binding targets, and are difficult to combine and adjust with various biotin-labeled substances, resulting in a complex functionalization process and relatively few supporting technologies for subsequent detection and analysis, which require further research and improvement. Summary of the Invention

[0004] The technical problem to be solved by this invention is that the current technology for specifically separating exosomes using the antigen-antibody specific binding immune reaction has a long antigen-antibody binding time, the binding and elution conditions are relatively strict, and the cost of preparing antibodies is high, which poses a challenge to large-scale production and preparation.

[0005] To address the problems of existing technologies, this invention provides an SA-Tim4 protein, its preparation method, and its application in exosome isolation. This method can efficiently prepare SA-Tim4, overcome the problem of high production costs in existing separation technologies, and break through the limitations of traditional exosome isolation technologies, such as time consumption, high requirements for equipment and reaction conditions, and difficulty in large-scale separation.

[0006] To achieve the above objectives, the technical solution of this invention is: an SA-Tim4 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1:

[0008] .

[0009] A method for preparing the above-mentioned SA-Tim4 protein, which is prepared by expression and purification using Pichia pastoris X-33 and Escherichia coli BL21.

[0010] A method for preparing SA-Tim4 functionalized magnetic nanoparticles includes the following steps:

[0011] (1) Preparation of magnetic nanoparticles (MNPs);

[0012] (2) Preparation of silica-functionalized magnetic nanoparticles MNP@Si;

[0013] (3) Preparation of amino-functionalized magnetic nanoparticles MNP@NH2;

[0014] (4) Preparation of biotin-functionalized magnetic nanoparticles MNP@Biotin;

[0015] (5) Preparation of SA-Tim4 functionalized magnetic nanoparticles MNP-Biotin@SA-Tim4.

[0016] Further, step (1) for preparing magnetic nanoparticles is as follows: 1.5g of ferric chloride hexahydrate, 3.5g of anhydrous sodium acetate, and 1mL of polyethyleneimine are dissolved in 40mL of ethylene glycol and stirred at room temperature for 2h with a rotor of 3cm and a rotation speed of 200r / min. When the drugs are completely dissolved and the mixture turns brownish-red, the mixture is poured into a 50mL polytetrafluoroethylene liner, and then the liner is placed in a high-pressure reactor, tightened, and placed in a muffle furnace. The reaction is carried out at 220℃ for 8h. After the reaction is completed, the high-pressure reactor is removed and cooled to ambient temperature at room temperature. The mixture is then poured into a 500mL beaker, and the nanoparticles are adsorbed by a strong magnet through the beaker wall. The nanoparticles are washed 6 times with anhydrous ethanol and ultrapure water, respectively. After the washing liquid is clear and transparent, the nanoparticles are stored in anhydrous ethanol and labeled as MNP.

[0017] Further, step (2) for preparing silica-functionalized magnetic nanoparticles is as follows: 100 mg of MNP magnetic beads are dispersed in 140 mL of ethanol-water solution (ethanol:water = 6:1, v / v), and ultrasonically dispersed for 40 min. Then, 2 mL of ammonia water (25% aqueous solution) is added. The mixture is poured into a 250 mL round-bottom flask and 20 glass beads (1 mm in diameter) are added. The round-bottom flask is fixed on a rotary evaporator and stirred at 180 r / min for 20 min at room temperature. During this process, 2 mL of tetraethoxysilane is added dropwise, and then stirred at 180 r / min for 3 h at room temperature. After the reaction is completed, the nanoparticles are adsorbed by a strong magnet through the flask wall and washed three times with anhydrous ethanol and ultrapure water, respectively. Then, the nanoparticles are stored in anhydrous ethanol and labeled as silica-functionalized magnetic beads MNP@Si.

[0018] Further, step (3) for preparing amino-functionalized magnetic nanoparticles is as follows: 450 mg of MNP@Si is ultrasonically dispersed in 150 mL of APTES-ethanol solution (4%, v / v); the mixture is poured into a 250 mL round-bottom flask and 20 glass beads (1 mm in diameter) are added. The round-bottom flask is fixed on a rotary evaporator and stirred at 180 r / min for 3 h at room temperature. After the reaction is completed, the nanoparticles are adsorbed by a strong magnet through the flask wall and washed three times with anhydrous ethanol and ultrapure water, respectively. Then the nanoparticles are stored in anhydrous ethanol and are referred to as amino-functionalized magnetic nanoparticles MNP@NH2.

[0019] Further, step (4) for preparing biotin-functionalized magnetic nanoparticles involves dissolving 240 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 200 mg of N-hydroxysuccinimide in 40 mL of PBS solution (0.01 M, pH 7.4). After stirring at room temperature for 20 min, 1 mL of biotin-PBS aqueous solution (1 mg / mL) is added, and the mixture is stirred at room temperature for 1 h. Then, 500 mg of MNP@NH2 is added, and the mixture is poured into a 50 mL centrifuge tube and stirred at 4 °C for 12 h. After the reaction is complete, the magnetic nanoparticles are adsorbed onto the wall of the centrifuge tube using a strong magnet, washed three times with PBS, and then stored in PBS. These are designated as biotin-functionalized magnetic nanoparticles MNP@Biotin.

[0020] Further, step (5) for preparing SA-Tim4-functionalized magnetic nanoparticles is as follows: 500 mg of MNP@Biotin is added to PBS containing SVA-Tim4, the mixture is poured into a 50 mL centrifuge tube, and stirred at 4 °C for 6 h; after the reaction is completed, the magnetic nanoparticles are isolated from the wall of the centrifuge tube by a strong magnet, washed 3 times with PBS, and then stored in PBS, which is named SA-Tim4-functionalized magnetic nanoparticles MNP-Biotin@SA-Tim4.

[0021] Application of the aforementioned SA-Tim4 protein or SA-Tim4-functionalized magnetic nanoparticles in the isolation of exosomes.

[0022] A method for separating and extracting exosomes using the aforementioned SA-Tim4 protein involves using SA-Tim4 combined with biotin-modified magnetic nanobeads to obtain SA-Tim4-functionalized magnetic nanoparticles. Exosome separation is achieved through the specific binding and release of Tim4 with phosphatidylserine residues on the surface of exosomes. The biotin-modified magnetic nanobeads can specifically bind SA-Tim4 from other proteins. SA-Tim4 is prepared by combining a recombinant protein of T cell immunoglobulin mucin 4 (Tim4) and streptavidin (SA) with biotin-modified magnetic nanobeads. The specific binding and release of Tim4 with phosphatidylserine residues on the surface of exosomes facilitates exosome separation. SA acts as a link between the affinity ligand Tim4 and the matrix MNP@Biotin in the exosome separation material. The binding site of biotin on SA is mainly composed of tryptophan residues from SA. This structural complementarity allows the biotin on MNP@Biotin to be precisely and easily embedded into the binding site of SA, thereby rapidly coupling MNP@Biotin and SA-Tim4 together in a physiological system to prepare an affinity medium for recognizing exosomes.

[0023] Further steps include: accurately weighing 0.2 g of SA-Tim4 functionalized magnetic nanoparticles and placing them into a 50 mL centrifuge tube. Washing MNP-Biotin@SA-Tim4 with 3 mL of PBS equilibration buffer (0.01 M, pH 7.4) three times, and then waiting for sample loading; adding 40 mL of sample extract filtered through a 0.22 μm filter to the centrifuge tube, and then shaking at 4 °C for 30 min; after adsorption, fixing MNP-Biotin@SA-Tim4 in the centrifuge tube with a strong magnet, and discarding the sample extract. Then adding 3 mL of PBS equilibration buffer, shaking for 1 min, fixing MNP-Biotin@SA-Tim4 in the centrifuge tube with a strong magnet, discarding the PBS equilibration buffer, and repeating twice to remove non-specifically bound exosomes. Finally, add 2 mL of elution buffer (PBS, 0.01 M, pH 7.4; EDTA-Na2, 2 mM), shake for 5 min, and then use a strong magnet to isolate the heart tube to fix MNP-Biotin@SA-Tim4. Take the elution buffer (which is rich in exosome particles) and store it at 4 °C.

[0024] The beneficial effects of this invention are as follows:

[0025] This invention utilizes engineered yeast and E. coli to prepare SA-Tim4, which specifically binds to biotin-modified magnetic nanobeads. Then, by leveraging the specific binding and separation of Tim4 with phosphatidylserine on the surface of exosomes, highly efficient separation of exosomes is achieved. Compared to methods such as ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, polymer precipitation, and microfluidics, this method eliminates reliance on equipment, removes the complexity of chemical reagent processes, and saves costs. It enables large-scale, green, environmentally friendly, and highly efficient preparation, possessing excellent potential for industrial applications. Attached Figure Description

[0026] Figure 1 Flowchart of the present invention for isolating exosomes

[0027] Figure 2 The diagram shows the target gene of SA-Tim4 expressed in Pichia pastoris in this invention, where M is the marker standard DNA.

[0028] Figure 3 : PCR verification diagram of the target gene of the recombinant plasmid pPICZαA-SA-Tim4 in this invention, where M is the marker standard DNA.

[0029] Figure 4 The image shows the double enzyme digestion verification of the recombinant plasmid pET-28a-SA-Tim4 in this invention, where M is the marker standard DNA.

[0030] Figure 5 The SDS-PAGE electrophoresis diagram of Pichia pastoris expression in this invention shows that M is the marker standard protein; 1 is the crude protein; and 2 is the purified target protein.

[0031] Figure 6 The image shows an SDS-PAGE electrophoresis diagram of SA-Tim4 expressed in Escherichia coli in this invention, where M is the marker standard protein; 1 is the supernatant of lysed bacteria without the target protein; 2 is the crude protein; 3 is the protein eluted with 5 mM imidazole; 4 is the protein eluted with 10 mM imidazole; 5 is the protein eluted with 20 mM imidazole; 6 is the protein eluted with 40 mM imidazole; 7 is the protein eluted with 100 mM imidazole; 8 is the protein eluted with 200 mM imidazole; and 9 is the protein eluted with 500 mM imidazole.

[0032] Figure 7 The diagram shows the comparison of the effects of biotin-modified magnetic nanobeads on SA-Tim4-containing heterogeneous proteins before and after their interaction. M represents the marker standard protein; 1 represents the SA-Tim4-containing heterogeneous protein; and 2 represents the protein after binding with biotin-modified magnetic nanobeads.

[0033] Figure 8 The following are SEM characterization results of magnetic nanoparticles at different modification stages in this invention: A represents magnetic nanoparticles (MNP); B represents silica-functionalized magnetic nanoparticles (MNP@Si); C represents amino-functionalized magnetic nanoparticles (MNP@NH2); D represents biotin-functionalized magnetic nanoparticles (MNP@Biotin); and E represents SA-Tim4-functionalized magnetic nanoparticles (MNP-Biotin@SA-Tim4).

[0034] Figure 9 The following are TEM characterization results of exosomes after adsorption and elution in this invention. A is MNP-Biotin@SA-Tim4 with adsorbed exosomes; B is MNP-Biotin@SA-Tim4 after desorption; and C is exosome vesicles after desorption. Detailed Implementation

[0035] The following description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0036] The following embodiments can be understood as illustrating only a part of the structure or method of the present invention, or as a combination of embodiments explaining the broader structure or method of the present invention. Unless otherwise specified, all materials used in the present invention were obtained through commercial channels.

[0037] Example 1:

[0038] An SA-Tim4 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0039] SEQ ID NO.1:

[0040] .

[0041] Example 2:

[0042] The construction and expression of the SA-Tim4 Pichia pastoris expression recombinant plasmid described in Example 1 included the following steps: The nucleotide sequence corresponding to SEQ ID NO.1 was submitted to Shanghai Sangon Biotech for optimization and synthesis. The upstream primer connected to the Pichia pastoris expression vector pPICZαA was 5'-GCTGAATTCATGTCCAAGGGACTCTTGCTTCTTTGGTTGGTCACC-3', and the downstream primer was 5'-GCTGGCGGCCGCTTGTTGAACAGCGTCC-3'. The PCR reaction conditions were: 95℃ pre-denaturation for 10 min, 98℃ denaturation for 10 s, 65℃ annealing for 30 s, 68℃ extension for 2 min, for 30 cycles, followed by a final extension at 65℃ for 10 min. The target gene cloning results are shown in [the original text]. Figure 2The target gene was ligated into the vector pPICZαA using the EcoRI and NotI restriction sites. The ligation product was transformed into E. coli DH5α competent cells, and sequencing was successful after positive verification. The recombinant plasmid was linearized with SacI and transformed into Pichia pastoris competent cells X-33. The cells were plated onto YPD solid medium plates containing bleomycin and incubated at 30°C for 48 h. Colonies were then selected for PCR positive verification. Figure 3 ), and sent to bioengineering for sequencing.

[0043] Identified positive clones were inoculated into 50 mL of BMGY medium and cultured at 30°C with shaking for 24 h. After the A600 reached 4.0–6.0, the culture was centrifuged at 3000 rpm for 5 min, and the precipitate was collected. The precipitate was resuspended in 30 mL of BMGY medium and cultured at 30°C with shaking to induce expression. Methanol was added every 24 h, maintaining a methanol concentration of 0.5%. After 7 days of methanol-induced culture, the culture was centrifuged at 12000 rpm for 10 min, and the supernatant was the crude protein.

[0044] Recombinant protein purification includes the following steps: Crude protein is purified by affinity chromatography using a Ni-NTA column. The column is equilibrated with 50 mM PB, 300 mM NaCl, 1% glycerol, and pH 8.0. The protein is then eluted with 5 mM, 10 mM, 20 mM, 40 mM, 100 mM, and 200 mM imidazole solutions (50 mM PB, 300 mM NaCl, 1% glycerol). The target protein is obtained by elution with 20 mM imidazole solution. The resulting solution is then analyzed by SDS-PAGE. Figure 5 and 6 Protein concentration was determined using the Bradford method.

[0045] Example 3:

[0046] The construction and expression of the SA-Tim4 E. coli expression recombinant plasmid described in Example 1 included the following steps: The nucleotide sequence corresponding to SEQ ID NO.1 was submitted to Shanghai Sangon Biotech for synthesis. The upstream primer ligated to the E. coli expression vector pET-28a(+) was 5'-CCGAATTCATGTCCAAGGGGCTTCTCCTCCTCTGGCTG-3', and the downstream primer was 5'-GTGCGGCCGCCTGCTGAACGGCGTC-3'. The PCR reaction conditions were: 95℃ pre-denaturation for 10 min, 98℃ denaturation for 10 s, 66℃ annealing for 30 s, 68℃ extension for 2 min, for 30 cycles, followed by a final extension at 68℃ for 10 min. The target gene was ligated to the vector pET-28a(+) using the EcoRI and NotI restriction enzyme sites. The ligation product was transformed into E. coli DH5α competent cells. The amplified recombinant plasmid was verified by double digestion with EcoRI and NotI. Figure 4 The sequencing was successful. The recombinant plasmid was transformed into the expression strain E. coli BL21(DE3), and cultured at 20℃ and 220 rpm for 48 h. The bacterial cells were then collected (centrifuged at 4℃ and 8000 rpm for 10 min). The bacterial cells were resuspended in 50 mM phosphate-buffered saline (PB) at pH 7.4, sonicated for 50 min, and centrifuged at 12000 rpm for 20 min. The supernatant was the crude protein.

[0047] Everything else is the same as in Example 2.

[0048] Example 4:

[0049] A method for preparing SA-Tim4 functionalized magnetic nanoparticles includes the following steps:

[0050] (1) The preparation method of magnetic nanoparticles is as follows: 3g of ferric chloride hexahydrate, 7g of anhydrous sodium acetate, and 1.5mL of polyethyleneimine were dissolved in 80mL of ethylene glycol and stirred at 26℃ for 2h with a rotor of 3cm and a rotation speed of 200r / min. When the reagents were completely dissolved and the mixture turned brownish-brown, the mixture was poured into a 100mL polytetrafluoroethylene liner, and then the liner was placed in a high-pressure reactor, tightened, and placed in a muffle furnace. The reaction was carried out at 220℃ for 8h. After the reaction was completed, the high-pressure reactor was removed and cooled to 26℃ at room temperature. The mixture was then poured into a 500mL beaker, and the nanoparticles were adsorbed by a strong magnet through the beaker wall. The nanoparticles were washed 6 times with anhydrous ethanol and ultrapure water, respectively. After the washing liquid was clear and transparent, the nanoparticles were vacuum dried and labeled as MNP.

[0051] (2) The preparation method of silica-functionalized magnetic nanoparticles is as follows: 0.2 g of MNP magnetic nanoparticles were dispersed in 280 mL of ethanol-water solution (ethanol:water = 6:1, v / v), and ultrasonically dispersed for 50 min. Then, 4 mL of ammonia water (25% aqueous solution) was added. The mixture was poured into a 300 mL round-bottom flask, and 25 glass beads (particle size 1 mm) were added. The round-bottom flask was fixed on a rotary evaporator and stirred at 200 r / min at 26 °C for 20 min. During this process, 4 mL of tetraethoxysilane was added dropwise, and then stirred at 200 r / min at 26 °C for 3 h. After the reaction was completed, the magnetic nanoparticles were adsorbed onto the flask wall with a strong magnet, washed three times with anhydrous ethanol and ultrapure water, respectively, and then vacuum dried. These were then labeled as silica-functionalized magnetic nanoparticles (MNP@Si).

[0052] (3) The preparation method of amino-functionalized magnetic nanoparticles is as follows: 0.2 g of MNP@Si was ultrasonically dispersed in 100 mL of APTES-ethanol solution (4%, v / v). The mixture was poured into a 250 mL round-bottom flask, and 12 glass beads (1 mm in diameter) were added. The round-bottom flask was fixed on a rotary evaporator and stirred at 200 r / min for 3 h at room temperature. After the reaction was completed, the nanoparticles were adsorbed onto the flask wall using a strong magnet. The nanoparticles were washed three times with anhydrous ethanol and ultrapure water, respectively. Then, the nanoparticles were stored in anhydrous ethanol and labeled as amino-functionalized magnetic nanoparticles (MNP@NH2).

[0053] (4) The preparation method of biotin-functionalized magnetic nanoparticles is as follows: 120 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 100 mg of N-hydroxysuccinimide were dissolved in 20 mL of PBS solution (0.01 M, pH 7.4). After stirring at room temperature for 20 min, 0.5 mL of biotin-PBS aqueous solution (1 mg / mL) was added. After stirring at room temperature for 1 h, 0.2 g of MNP@NH2 was added. The mixture was poured into a 50 mL centrifuge tube and stirred at 4 °C for 10 h. After the reaction was completed, the magnetic nanoparticles were adsorbed onto the wall of the centrifuge tube using a strong magnet. The tubes were washed three times with PBS and then stored in PBS, which were designated as biotin-functionalized magnetic nanoparticles (MNP@Biotin).

[0054] SA-Tim4 can specifically adsorb onto biotin-modified magnetic beads (MNP@Biotin), including the following steps: After SA-Tim4 protein containing impure bands expressed in E. coli BL21 binds to biotin-modified magnetic nanobeads, the disappearance of the target band can be clearly observed. Figure 7 This demonstrates that biotin-modified magnetic nanobeads can specifically bind to SA-Tim4.

[0055] (5) The preparation method of SA-Tim4 functionalized magnetic nanoparticles is as follows: 500 mg of MNP@Biotin was added to PBS containing SA-Tim4, and the mixture was poured into a 50 mL centrifuge tube and stirred at 4 °C for 6 h. After the reaction was completed, the magnetic nanoparticles were adsorbed onto the wall of the centrifuge tube using a strong magnet, washed three times with PBS, and then stored in PBS, which were designated as SA-Tim4 functionalized magnetic nanoparticles (MNP-Biotin@SA-Tim4).

[0056] Example 5:

[0057] A method for purifying exosomes from watermelon extract using MNP-Biotin@SA-Tim4 as described in Example 9 includes the following steps: The watermelon is peeled, the pulp is extracted, and juiced. The juice is filtered through a 50 μm sieve to remove pulp and other impurities. The coarsely filtered watermelon juice is centrifuged at 800 rpm for 15 min at 4°C to remove free watermelon cells. The supernatant is then centrifuged again at 9000 rpm for 15 min at 4°C to remove broken watermelon cell fragments. Finally, the supernatant is filtered through a 0.22 μm filter into a new container for later use.

[0058] Accurately weigh 0.3 g of MNP-Biotin@SA-Tim4 into a 50 mL centrifuge tube. Wash MNP-Biotin@SA-Tim4 with 3 mL of PBS equilibration buffer (0.01 M, pH 7.4), repeating 3 times, and wait for sample loading. Add 40 mL of watermelon extract filtered through a 0.22 μm filter to the centrifuge tube, and then shake at 4 °C for 30 min. After adsorption, fix MNP-Biotin@SA-Tim4 in the centrifuge tube using a strong magnet, and discard the kelp extract. Then add 3 mL of PBS equilibration buffer, shake for 1 min, fix MNP-Biotin@SA-Tim4 in the centrifuge tube using a strong magnet, discard the PBS equilibration buffer, and repeat twice to remove non-specifically bound exosomes. Finally, add 2 mL of elution buffer (PBS, 0.01 M, pH 7.4; EDTA-Na2, 2 mM), shake for 5 min, and then fix MNP-Biotin@SA-Tim4 in a cardiopulmonary bypass tube using a strong magnet. Collect the eluent (which is rich in exosome particles) and store it at 4°C. Send the eluent to a TEM to determine if it contains exosome particles.

[0059] Material characterization:

[0060] (1) SEM characterization results of functionalized magnetic nanoparticles

[0061] The results showed that modifying the magnetic nanoparticles with silica altered their surface activity, making them more cohesive and causing the magnetic beads to aggregate into clusters. Subsequent modifications with amino groups, biotin, and SA-Tim4 did not significantly change their appearance.

[0062] (2) TEM characterization results of MNP-Biotin@SA-Tim4 purified exosomes

[0063] The results showed that after adsorbing exosomes, and after rinsing away impurities and non-specifically bound exosomes with buffer solution, exosome vesicles were bound to the surface of the magnetic beads. Figure 9(A) This indicates that MNP-Biotin@SA-Tim4 exhibits excellent adsorption performance on exosomes. After elution with eluent, no exosome vesicles were found on the surface of MNP-Biotin@SA-Tim4. Figure 9 B), obvious exosome vesicles could be observed in the eluent. Figure 9 (C) This indicates that MNP-Biotin@SA-Tim4 has a significant enrichment effect on exosomes, and the application of this patent in actual samples is feasible.

[0064] Example 6:

[0065] Sample preparation: The kelp treatment solution was prepared by crushing kelp and dissolving it in PBS solution (0.01M, pH 7.4), centrifuging at 800 r / min for 15 min at 6℃ to remove free kelp cells from the sample; the supernatant was then centrifuged at 9000 r / min for 15 min at 6℃ to remove broken kelp cell fragments from the sample; finally, the supernatant was filtered through a 0.22 μm filter into a new container for later use.

[0066] Sample preparation: Accurately weigh 0.2 g of SA-Tim4 functionalized magnetic nanoparticles and place them in a 50 mL centrifuge tube. Wash MNP-Biotin@SA-Tim4 with 3 mL of PBS equilibration buffer (0.01 M, pH 7.4), repeating 3 times, and wait for sample loading. Add 40 mL of kelp extract filtered through a 0.22 μm filter to the centrifuge tube, and then shake at 4 °C for 30 min. After adsorption, use a strong magnet to isolate the centrifuge tube to fix MNP-Biotin@SA-Tim4, and discard the kelp extract. Then add 3 mL of PBS equilibration buffer, shake for 1 min, and use a strong magnet to isolate the centrifuge tube to fix MNP-Biotin@SA-Tim4, discard the PBS equilibration buffer, and repeat twice to remove non-specifically bound exosomes. Finally, add 2 mL of elution buffer (PBS, 0.01 M, pH 7.4; EDTA-Na2, 2 mM), shake for 5 min, and then use a strong magnet to isolate the heart tube to fix MNP-Biotin@SA-Tim4. Take the elution buffer (which is rich in exosome particles) and store it at 4 °C.

[0067] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0068] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An SA-Tim4 protein, characterized in that: The amino acid sequence is shown in SEQ ID NO.1; it was prepared by expression and purification using Pichia pastoris X-33 and Escherichia coli BL21.

2. An SA-Tim4 functionalized magnetic nanoparticle, the preparation method of which includes the following steps: (1) Preparation of magnetic nanoparticles (MNPs); (2) Preparation of silica-functionalized magnetic nanoparticles MNP@Si; (3) Preparation of amino-functionalized magnetic nanoparticles MNP@NH2; (4) Preparation of biotin-functionalized magnetic nanoparticles MNP@Biotin; (5) Preparation of SA-Tim4 functionalized magnetic nanoparticles MNP-Biotin@SA-Tim4; The amino acid sequence of the SA-Tim4 protein is shown in SEQ ID NO.

1.

3. The particles as described in claim 2, characterized in that: Step (1) Preparation of magnetic nanoparticles: Ferric chloride hexahydrate, anhydrous sodium acetate, and polyethyleneimine are dissolved in ethylene glycol and stirred at room temperature; when the drugs are completely dissolved and the mixture turns brownish-red, the mixture is poured into a polytetrafluoroethylene liner and reacted at 220°C for 8 h; after the reaction is completed, the mixture is cooled to ambient temperature at room temperature, and then the mixture is poured into a beaker. The nanoparticles are adsorbed by a strong magnet through the beaker wall, and washed with anhydrous ethanol and ultrapure water respectively. After the washing solution is clear and transparent, the nanoparticles are stored in anhydrous ethanol and labeled as MNP.

4. The particles as described in claim 2, characterized in that: Step (2) to prepare silica-functionalized magnetic nanoparticles is as follows: MNP magnetic beads are dispersed in an ethanol aqueous solution, ultrasonically dispersed, and then ammonia is added; the mixture is poured into a flask and glass beads are added. The flask is fixed on a rotary evaporator and stirred at room temperature. During this process, 2 mL of tetraethoxysilane is added dropwise and then stirred at room temperature; after the reaction is completed, the nanoparticles are adsorbed by a strong magnet through the flask wall, washed with anhydrous ethanol and ultrapure water respectively, and then stored in anhydrous ethanol, which is denoted as silica-functionalized magnetic beads MNP@Si.

5. The particles as described in claim 2, characterized in that: Step (3) Preparation of amino-functionalized magnetic nanoparticles: MNP@Si was ultrasonically dispersed in APTES-ethanol solution; the mixture was poured into a flask and a glass bead was added. The flask was fixed on a rotary evaporator and stirred at room temperature; after the reaction was completed, the nanoparticles were adsorbed by a strong magnet through the flask wall, washed with anhydrous ethanol and ultrapure water respectively, and then stored in anhydrous ethanol, which was denoted as amino-functionalized magnetic nanoparticles MNP@NH2.

6. The particles as described in claim 2, characterized in that: Step (4) Preparation of biotin-functionalized magnetic nanoparticles: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in PBS solution and stirred at room temperature. Biotin PBS aqueous solution was added and stirred at room temperature. MNP@NH2 was added and the mixture was poured into a centrifuge tube and stirred at 4°C. After the reaction was completed, the magnetic nanoparticles were isolated from the wall of the centrifuge tube by a strong magnet, washed with PBS, and then stored in PBS. These were designated as biotin-functionalized magnetic nanoparticles MNP@Biotin.

7. The particles as described in claim 2, characterized in that: Step (5) Preparation of SA-Tim4-functionalized magnetic nanoparticles: MNP@Biotin was added to PBS containing SA-Tim4, the mixture was poured into a centrifuge tube and stirred at 4°C; after the reaction was completed, the magnetic nanoparticles were isolated from the wall of the centrifuge tube by a strong magnet, washed with PBS, and then stored in PBS, which were denoted as SA-Tim4-functionalized magnetic nanoparticles MNP-Biotin@SA-Tim4.

8. The application of the SA-Tim4 functionalized magnetic nanoparticles according to any one of claims 2-7 in the separation of exosomes.

9. A method for separating and extracting exosomes using SA-Tim4 functionalized magnetic nanoparticles as described in any one of 2-7, characterized in that: SA-Tim4-functionalized magnetic nanoparticles were obtained by modifying nanoparticles with biotin using SA-Tim4. Exosomes were then separated by the specific binding and release of phosphatidylserine on the surface of exosomes using Tim4.

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