SA-Tim4 protein, preparation method thereof and application of SA-Tim4 protein in exosome separation

Abstract

The invention belongs to the technical field of recombinant proteins and applications thereof, and particularly relates to an SA-Tim4 protein, a preparation method thereof, and applications of the SA-Tim4 protein in exosome separation. The amino acid sequence of the gene is as shown in SEQ ID NO. 1. The invention provides SA-Tim4 protein, a preparation method thereof and application of the SA-Tim4 protein in exosome separation, SA-Tim4 can be efficiently prepared, the problem of high production cost of an existing separation technology is solved, and the limitation that a traditional exosome separation technology is time-consuming, high in equipment and reaction condition requirements and difficult in large-scale separation is broken through.

Description

Technical Field

[0001] The present invention belongs to the technical field of recombinant proteins and their applications, and specifically relates to a SA-Tim4 protein and a preparation method thereof and an application thereof in isolating exosomes. Background Art

[0002] Exosomes are extracellular vesicles formed by cells through autocrine or paracrine means, with a diameter of about 30 to 150 nm. They are rich in DNA, RNA, proteins, etc. that participate in cell signal transduction and regulate 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 separation technology is the key to restricting the widespread application of exosomes, and efficient, portable, high-purity, and large-scale preparation technology has become a research hotspot. Existing exosome separation technologies mainly focus on separation using density differences, density differences, solubility, and fluid properties, which have problems such as low separation purity, high equipment requirements, and long time consumption. In contrast, the affinity separation technology of immune response can specifically separate exosomes using the immune response of antigen-antibody specific binding. Chinese patent CN113046303A "A rapid extraction kit for exosome separation" and Chinese patent CN111849903A "A kit for separating exosomes from cell supernatant and its use method" both mention the use of TIMD4 recombinant protein, TIMD3 recombinant protein, or AnnexinV recombinant protein as components for recognizing and binding exosomes. However, the relative specificity of TIMD4 recombinant protein, TIMD3 recombinant protein or AnnexinV recombinant protein is insufficient, and their binding targets are relatively fixed, making it difficult to conveniently combine and adjust with various biotin-labeled substances, resulting in a complex functional modification process. There are relatively few supporting technologies for subsequent detection and analysis, which requires further research and improvement. Summary of the invention

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

[0005] In view of the problems of the prior art, the present invention provides a SA-Tim4 protein and a preparation method thereof and an application in isolating exosomes, which can efficiently prepare SA-Tim4, overcome the problem of high production cost of existing separation technology, and break the limitations of traditional exosome separation technology, which is time-consuming, requires high equipment and reaction conditions, and is difficult to separate on a large scale.

[0006] To achieve the above-mentioned purpose, the technical solution of the present invention is: a SA-Tim4 protein, whose amino acid sequence is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1:

[0008] .

[0009] A method for preparing the SA-Tim4 protein is to express, purify and prepare it using Pichia pastoris X-33 and Escherichia coli BL21.

[0010] A SA-Tim4 functionalized magnetic nanoparticle, the preparation method of which comprises the following steps:

[0011] (1) preparing magnetic nanoparticles MNP;

[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] Furthermore, the step (1) of preparing magnetic nanoparticles is as follows: 1.5 g of ferric chloride hexahydrate, 3.5 g of anhydrous sodium acetate, and 1 mL of polyethyleneimine are dissolved in 40 mL of ethylene glycol, and stirred at room temperature for 2 h, with a rotor of 3 cm and a rotation speed of 200 r / min; when the drug is completely dissolved and the mixed solution appears brown, the mixed solution is poured into a 50 mL polytetrafluoroethylene liner, and then the liner is placed in a high-pressure reactor, and after being tightened, it is placed in a muffle furnace and reacted at 220° C. for 8 h; after the reaction is completed, the high-pressure reactor is taken out, cooled to ambient temperature at room temperature, and then the mixed solution is poured into a 500 mL beaker, and the nanomagnetic beads are adsorbed through the beaker wall with a strong magnet, and washed with anhydrous ethanol and ultrapure water for 6 times respectively. After the washing liquid becomes clear and transparent, the nanomagnetic beads are stored in anhydrous ethanol, which is recorded as MNP.

[0017] Furthermore, step (2) is to prepare silica-functionalized magnetic nanoparticles as follows: 100 mg of MNP magnetic beads are dispersed in 140 mL of ethanol aqueous solution (ethanol: water = 6: 1, v / v), ultrasonically dispersed for 40 min, and then 2 mL of ammonia water (25% aqueous solution) is added; the mixed solution is poured into a 250 mL round-bottom flask, and 20 glass beads (particle size 1 mm) 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 which 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 nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, and washed three times with anhydrous ethanol and ultrapure water respectively, and then the nanomagnetic beads are stored in anhydrous ethanol, which is recorded as silica-functionalized magnetic beads MNP@Si.

[0018] Furthermore, step (3) is to prepare amino-functionalized magnetic nanoparticles by ultrasonically dispersing 450 mg of MNP@Si in 150 mL of APTES-ethanol solution (4%, v / v); pouring the mixture into a 250 mL round-bottom flask, adding 20 glass beads (particle size 1 mm), fixing the round-bottom flask on a rotary evaporator, and stirring at 180 r / min for 3 hours at room temperature; after the reaction is completed, using a strong magnet to adsorb the nanomagnetic beads through the flask wall, washing them with anhydrous ethanol and ultrapure water for 3 times respectively, and then storing the nanomagnetic beads in anhydrous ethanol, which are recorded as amino-functionalized magnetic nanoparticles MNP@NH2.

[0019] Furthermore, step (4) is to prepare biotin functionalized magnetic nanoparticles by 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), stirring at room temperature for 20 min, adding 1 mL of biotin PBS aqueous solution (1 mg / mL), stirring at room temperature for 1 h, adding 500 mg of MNP@NH2, pouring the mixture into a 50 mL centrifuge tube, and stirring at 4°C for 12 h. After the reaction is completed, the nanomagnetic beads are adsorbed on the heart tube wall with a strong magnet, washed with PBS 3 times, and then stored in PBS, which is recorded as biotin functionalized magnetic nanoparticles MNP@Biotin.

[0020] Furthermore, step (5) is to prepare SA-Tim4 functionalized magnetic nanoparticles by adding 500 mg of MNP@Biotin to PBS containing SVA-Tim4, pouring the mixture into a 50 mL centrifuge tube, and stirring at 4°C for 6 hours; after the reaction is completed, using a strong magnet to isolate the nanomagnetic beads adsorbed by the heart tube wall, washing with PBS for 3 times, and then storing the nanomagnetic beads in PBS, which are recorded as SA-Tim4 functionalized magnetic nanoparticles MNP-Biotin@SA-Tim4.

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

[0022] A method for separating and extracting exosomes using the above-mentioned SA-Tim4 protein, using SA-Tim4 combined with biotin-modified nanomagnetic beads to obtain SA-Tim4 functionalized magnetic nanoparticles, and separating exosomes by specific binding and release of Tim4 with phosphatidylserine on the surface of exosomes. The biotin-modified nanomagnetic beads can specifically bind SA-Tim4 from miscellaneous proteins. SA-Tim4 is prepared by combining a recombinant protein composed of T cell immunoglobulin mucin 4 (Tim4) and streptavidin (SA) with biotin-modified nanomagnetic beads, and exosomes are separated by specific binding and release of Tim4 with phosphatidylserine on the surface of exosomes. SA plays a role in connecting the affinity ligand Tim4 and the matrix MNP@Biotin in the exosome separation material, and the binding site of biotin on SA is mainly composed of tryptophan residues of SA. This structural complementarity enables the Biotin on MNP@Biotin to be accurately and easily embedded into the binding site of SA, thereby quickly coupling MNP@Biotin and SA-Tim4 under a physiological system to prepare an affinity medium for identifying exosomes.

[0023] Further, the following steps are included: accurately weigh 0.2g of SA-Tim4 functionalized magnetic nanoparticles and put them into a 50mL centrifuge tube. Wash MNP-Biotin@SA-Tim4 with 3mL PBS balance solution (0.01M, pH 7.4), repeat 3 times, and wait for sample loading; add 40mL of sample extract filtered through a 0.22μm filter into a centrifuge tube, and then shake at 4°C for 30min; after adsorption, use a strong magnet to isolate the heart tube to fix MNP-Biotin@SA-Tim4, and pour out the sample extract. Then add 3mL PBS balance solution, shake for 1min, use a strong magnet to isolate the heart tube to fix MNP-Biotin@SA-Tim4, pour out the PBS balance solution, and repeat 2 times to remove non-specifically bound exosomes. Finally, 2 mL of elution buffer (PBS, 0.01 M, pH 7.4; EDTA-Na2, 2 mM) was added, and after shaking for 5 min, the MNP-Biotin@SA-Tim4 was fixed by isolating the core tube with a strong magnet, and the eluate (which was rich in exosome particles) was taken and stored at 4°C.

[0024] The beneficial effects of the present invention are:

[0025] The present invention uses yeast and Escherichia coli to prepare SA-Tim4 that can specifically bind to biotin-modified nanomagnetic beads, and then uses Tim4 to specifically bind and separate phosphatidylserine on the surface of exosomes, thereby achieving efficient separation of exosomes. Compared with ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, polymer precipitation and microfluidics, it eliminates the dependence on equipment, removes the cumbersomeness of chemical reagent processes and saves costs, and can achieve large-scale green, environmentally friendly and efficient preparation, with good industrial application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 :Flow chart of isolating exosomes of the present invention

[0027] Figure 2 : The target gene map of SA-Tim4 expressed in Pichia pastoris in the present invention, wherein M is the Marker standard DNA.

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

[0029] Figure 4 : The double enzyme digestion verification diagram of the recombinant plasmid pET-28a-SA-Tim4 in the present invention, wherein M is the Marker standard DNA.

[0030] Figure 5 : SDS-PAGE electrophoresis diagram of Pichia pastoris expression in the present invention, wherein M is the Marker standard protein; 1 is the crude protein; and 2 is the purified target protein.

[0031] Figure 6 : The SDS-PAGE electrophoresis diagram of SA-Tim4 expressed in Escherichia coli of the present invention, wherein M is the Marker standard protein; 1 is the supernatant of broken bacteria without the target protein; 2 is the crude protein; 3 is the protein eluted with 5mM imidazole; 4 is the protein eluted with 10mM imidazole; 5 is the protein eluted with 20mM imidazole; 6 is the protein eluted with 40mM imidazole; 7 is the protein eluted with 100mM imidazole; 8 is the protein eluted with 200mM imidazole; 9 is the protein eluted with 500mM imidazole.

[0032] Figure 7 : Comparison diagram of the biotin-modified nanomagnetic beads before and after acting on SA-Tim4-containing impurity proteins in the present invention, wherein M is the Marker standard protein; 1 is the impurity protein containing SA-Tim4; 2 is the protein after biotin-modified nanomagnetic beads bind.

[0033] Figure 8 : SEM characterization results of magnetic nanoparticles at different modification stages in the present invention, wherein A is magnetic nanoparticles (MNP); B is silica-functionalized magnetic nanoparticles (MNP@Si); C is amino-functionalized magnetic nanoparticles (MNP@NH2); D is biotin-functionalized magnetic nanoparticles (MNP@Biotin); E is SA-Tim4-functionalized magnetic nanoparticles (MNP-Biotin@SA-Tim4).

[0034] Fig. 9 : TEM characterization result diagram after adsorption and elution of exosomes in the present invention, wherein A is MNP-Biotin@SA-Tim4 adsorbed with exosomes; B is MNP-Biotin@SA-Tim4 after desorption; C is exosome vesicles after desorption. DETAILED DESCRIPTION

[0035] The following description is only 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 in the scope of protection of the present invention.

[0036] The following examples can be understood as a part of the local structure or method of the present invention, or can be understood as a combination of the examples to explain the connotation of the larger structure or method of the present invention. Unless otherwise specified, all raw materials of the present invention are purchased from commercial channels.

[0037] Embodiment 1:

[0038] A SA-Tim4 protein, whose amino acid sequence is shown in SEQ ID NO.1.

[0039] SEQ ID NO.1:

[0040] .

[0041] Embodiment 2:

[0042] The construction and expression of the SA-Tim4 Pichia pastoris expression recombinant plasmid described in Example 1 includes the following steps: submitting the nucleotide sequence corresponding to SEQ ID NO.1 to Shanghai Bioengineering for optimization and synthesis, the upstream primer connected to the Pichia pastoris expression vector pPICZαA: 5'-GCTGAATTCATGTCCAAGGGACTCTTGCTTCTTTGGTTGGTCACC-3', the downstream primer: 5'-GCTGGCGGCCGCTTGTTGAACAGCGTCC-3', and the PCR reaction conditions are: 95°C pre-denaturation for 10 min, 98°C denaturation for 10 s, 65°C annealing for 30 s, 68°C extension for 2 min, reaction for 30 cycles, and 65°C post-extension for 10 min. The target gene cloning results are shown in Figure 2, the target gene was connected to the vector pPICZαA using restriction sites EcoRI and NotI, and the ligation product was transferred into E.coli DH5α competent cells. After positive verification, sequencing was successful. The recombinant plasmid was linearized with SacI and transferred into Pichia pastoris competent cells X-33, and plated onto a YPD solid medium plate containing bleomycin. After culturing at 30°C for 48 hours, colonies were selected for PCR positive verification ( Figure 3 ) and sent to Bioinformatics for sequencing.

[0043] Take the identified positive clone strain, inoculate it in 50mL BMGY medium, shake culture at 30℃ for 24h, centrifuge at 3000r / min for 5min until A600 reaches 4.0-6.0, and collect the precipitate. Suspend it in 30mL BMMY culture medium, shake culture at 30℃, and induce expression. Add methanol every 24h, and the methanol concentration is always kept at 0.5%. After 7d of methanol induction culture, centrifuge at 12000r / min for 10min, and the supernatant is the crude protein.

[0044] The recombinant protein purification comprises the following steps: the crude protein is purified by affinity chromatography using a Ni-NTA column, the column is balanced using 50 mM PB, 300 mM NaCl, 1% glycerol, pH 8.0, and the protein is eluted using 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 eluted using 20 mM imidazole solution, and the obtained solution is subjected to SDS-PAGE detection ( Figure 5 and 6 ), and the protein concentration was determined using the Bradford method.

[0045] Embodiment 3:

[0046] The construction and expression of the SA-Tim4 E. coli expression recombinant plasmid described in Example 1 includes the following steps: submitting the nucleotide sequence corresponding to SEQ ID NO.1 to Shanghai Bio-Tech for synthesis, the upstream primer connected to the E. coli expression vector pET-28a(+): 5'-CCGAATTCATGTCCAAGGGGCTTCTCCTCCTCTGGCTG-3', the downstream primer: 5'-GTGCGGCCGCCTGCTGAACGGCGTC-3', and the PCR reaction conditions are: 95°C pre-denaturation for 10 min, 98°C denaturation for 10 s, 66°C annealing for 30 s, 68°C extension for 2 min, reaction for 30 cycles, and 68°C post-extension for 10 min. The target gene was connected to the vector pET-28a(+) using the restriction sites EcoRI and NotI, the connection product was transferred into E. coli DH5α competent cells, and the amplified recombinant plasmid was double-digested with EcoRI and NotI for verification ( Figure 4 ), and sent to Bioengineering for sequencing. The recombinant plasmid was transferred into the expression strain E. coli BL21 (DE3), cultured at 20°C, 220r / min, and collected after 48h (4°C, 8000r / min centrifugation for 10min). The cells were resuspended in 50mM pH 7.4 phosphate buffer (PB), ultrasonically disrupted for 50min, and centrifuged at 12000r / min for 20min. The supernatant was the crude protein.

[0047] The rest are the same as in Example 2.

[0048] Embodiment 4:

[0049] A SA-Tim4 functionalized magnetic nanoparticle, the preparation method of which comprises 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 are dissolved in 80mL of ethylene glycol, and stirred at 26°C for 2h, with a rotor of 3cm and a speed of 200r / min. When the drug is completely dissolved and the mixed solution appears brown, the mixed solution is poured into a 100mL polytetrafluoroethylene liner, and then the liner is placed in a high-pressure reactor, tightened and placed in a muffle furnace, and reacted at 220°C for 8h. After the reaction is completed, the high-pressure reactor is taken out and cooled to 26°C at room temperature, and then the mixed solution is poured into a 500mL beaker, and the nanomagnetic beads are adsorbed through the beaker wall with a strong magnet, and washed with anhydrous ethanol and ultrapure water for 6 times respectively. After the washing liquid becomes clear and transparent, the nanomagnetic beads are vacuum dried and recorded as MNP.

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

[0052] (3) The preparation method of amino-functionalized magnetic nanoparticles is as follows: 0.2 g of MNP@Si is ultrasonically dispersed in 100 mL of APTES-ethanol solution (4%, v / v). The mixture is poured into a 250 mL round-bottom flask, and 12 glass beads (particle size 1 mm) are added. The round-bottom flask is fixed on a rotary evaporator and stirred at 200 r / min for 3 h at room temperature. After the reaction is completed, the nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, washed three times with anhydrous ethanol and ultrapure water respectively, and then stored in anhydrous ethanol, which is recorded 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), stirred at room temperature for 20 min, and then 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, and the mixture was poured into a 50 mL centrifuge tube and stirred at 4°C for 10 h. After the reaction was completed, the nanomagnetic beads were isolated by a strong magnet to adsorb the nanoparticles on the wall of the heart tube, washed with PBS three times, and then stored in PBS, which was recorded as biotin functionalized magnetic nanoparticles (MNP@Biotin).

[0054] SA-Tim4 can be specifically adsorbed onto biotin-modified magnetic beads (MNP@Biotin), which includes the following steps: After the SA-Tim4 protein containing the impurity band expressed by E. coli BL21 is combined with the biotin-modified nanomagnetic beads, the disappearance of the target band can be clearly observed. Figure 7 , proving that biotin-modified nanomagnetic beads 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, the mixture was poured into a 50 mL centrifuge tube, and stirred at 4°C for 6 h. After the reaction, a strong magnet was used to isolate the nanomagnetic beads adsorbed on the heart tube wall, and the nanomagnetic beads were washed 3 times with PBS, and then stored in PBS, which was recorded as SA-Tim4 functionalized magnetic nanoparticles (MNP-Biotin@SA-Tim4).

[0056] Embodiment 5:

[0057] A method for purifying exosomes in a watermelon extract using MNP-Biotin@SA-Tim4 in Example 9 comprises the following steps: peeling the watermelon and extracting the pulp for juicing, filtering the juice with a 50 μm mesh to remove impurities such as the pulp. Centrifuging the coarsely filtered watermelon juice at 800 r / min for 15 min at 4°C to remove free watermelon cells in the sample; taking the supernatant, and then centrifuging it at 9000 r / min for 15 min at 4°C to remove broken watermelon cell fragments in the sample; finally, taking the supernatant and filtering it with a 0.22 μm filter into a new container for later use.

[0058] Accurately weigh 0.3g of MNP-Biotin@SA-Tim4 and put it into a 50mL centrifuge tube. Wash MNP-Biotin@SA-Tim4 with 3mL PBS balance solution (0.01M, pH 7.4), repeat 3 times, and wait for loading. Add 40mL of watermelon extract filtered through a 0.22μm filter to the centrifuge tube, and then shake at 4℃ for 30min. After the adsorption is completed, use a strong magnet to isolate the heart tube to fix MNP-Biotin@SA-Tim4, and pour out the kelp extract. Then add 3mL PBS balance solution, shake for 1min, use a strong magnet to isolate the heart tube to fix MNP-Biotin@SA-Tim4, pour out the PBS balance solution, and repeat 2 times to remove non-specifically bound exosomes. Finally, add 2 mL of eluent (PBS, 0.01 M, pH 7.4; EDTA-Na2, 2 mM), shake for 5 min, isolate the tube with a strong magnet to fix MNP-Biotin@SA-Tim4, take the eluent (the eluent is rich in exosome particles) and store it at 4 ° C. Send the eluent to TEM to determine whether it contains exosome particles.

[0059] Material Characterization:

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

[0061] The results showed that after the magnetic nanoparticles were modified with silica, the surface activity of the magnetic nanoparticles was changed, making them more cohesive and the magnetic beads aggregated into clusters. After the subsequent modification with amino groups, biotin and SA-Tim4, their appearance did not change significantly.

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

[0063] The results showed that after adsorbing exosomes, the impurities and non-specific binding exosomes were washed away by buffer solution, and exosome vesicles were bound to the surface of magnetic beads ( Fig. 9, A), which shows that MNP-Biotin@SA-Tim4 has an excellent adsorption effect on exosomes. After elution with the eluent, there are no more exosome vesicles on the surface of MNP-Biotin@SA-Tim4 ( Fig. 9 , B), obvious exosome vesicles can be observed in the eluate ( Fig. 9 , C), which shows that the enrichment effect of exosomes by MNP-Biotin@SA-Tim4 is significant, and the applicability of this patent in actual samples is feasible.

[0064] Embodiment 6:

[0065] Sample preparation: The kelp processing liquid is prepared by crushing the kelp, dissolving it in a PBS solution (0.01M, pH 7.4), and centrifuging it at 800r / min at 6°C for 15min to remove the free kelp cells in the sample; taking the supernatant, and then centrifuging it at 9000r / min at 6°C for 15min to remove the broken kelp cell fragments in the sample; finally, taking the supernatant and filtering it with a 0.22μm filter into a new container for later use.

[0066] Sample processing: Accurately weigh 0.2g of SA-Tim4 functionalized magnetic nanoparticles into a 50mL centrifuge tube. Wash MNP-Biotin@SA-Tim4 with 3mL PBS balance solution (0.01M, pH 7.4), repeat 3 times, and wait for sample loading. Add 40mL of kelp extract filtered through a 0.22μm filter into a centrifuge tube and shake at 4°C for 30min. After adsorption, use a strong magnet to isolate the heart tube to fix MNP-Biotin@SA-Tim4, and pour out the kelp extract. Then add 3mL PBS balance solution, shake for 1min, use a strong magnet to isolate the heart tube to fix MNP-Biotin@SA-Tim4, pour out the PBS balance solution, and repeat 2 times 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, use a strong magnet to isolate the heart tube to fix MNP-Biotin@SA-Tim4, take the eluate (the eluate is rich in exosome particles) and store it at 4°C.

[0067] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0068] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A SA-Tim4 protein, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing the SA-Tim4 protein according to claim 1, characterized in that: The protein was expressed and purified in Pichia pastoris X-33 and Escherichia coli BL21.

3. A SA-Tim4 functionalized magnetic nanoparticle, the preparation method of which comprises the following steps: (1) preparing magnetic nanoparticles MNP; (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.

4. The particle according to claim 3, characterized in that: The step (1) of preparing magnetic nanoparticles comprises: dissolving ferric chloride hexahydrate, anhydrous sodium acetate and polyethyleneimine in ethylene glycol and stirring at room temperature; when the drugs are completely dissolved and the mixed solution appears brown, pouring the mixed solution into a polytetrafluoroethylene liner and reacting at 220° C. for 8 hours; after the reaction is completed, cooling the mixture to ambient temperature at room temperature, then pouring the mixed solution into a beaker, using a strong magnet to adsorb the nanomagnetic beads through the beaker wall, washing with anhydrous ethanol and ultrapure water respectively, and storing the nanomagnetic beads in anhydrous ethanol after the washing solution becomes clear and transparent, and recording them as MNPs.

5. The particle according to claim 3, characterized in that: Step (2) of preparing silica-functionalized magnetic nanoparticles is as follows: dispersing MNP magnetic beads in an ethanol aqueous solution, ultrasonically dispersing the beads, and then adding ammonia water; pouring the mixed solution into a flask, adding glass beads, fixing the flask on a rotary evaporator, stirring at room temperature, and adding 2 mL of tetraethoxysilane dropwise during the process, and then stirring at room temperature; after the reaction is completed, using a strong magnet to adsorb the nanomagnetic beads through the wall of the flask, washing them with anhydrous ethanol and ultrapure water respectively, and then storing the nanomagnetic beads in anhydrous ethanol, which is recorded as silica-functionalized magnetic beads MNP@Si.

6. The particle according to claim 3, characterized in that: Step (3) is to prepare amino-functionalized magnetic nanoparticles by ultrasonically dispersing MNP@Si in an APTES-ethanol solution; pouring the mixed solution into a flask, adding glass beads, fixing the flask on a rotary evaporator, and stirring at room temperature; after the reaction is completed, using a strong magnet to adsorb the nanomagnetic beads through the wall of the flask, washing them with anhydrous ethanol and ultrapure water respectively, and then storing the nanomagnetic beads in anhydrous ethanol, which are recorded as amino-functionalized magnetic nanoparticles MNP@NH2.

7. The particle according to claim 3, characterized in that: Step (4) is to prepare biotin functionalized magnetic nanoparticles by dissolving 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in PBS solution, stirring at room temperature, adding biotin PBS aqueous solution, stirring at room temperature, adding MNP@NH2, pouring the mixture into a centrifuge tube, and stirring at 4°C; after the reaction is completed, using a strong magnet to isolate the nanomagnetic beads adsorbed by the heart tube wall, washing with PBS, and then storing the nanomagnetic beads in PBS, which are recorded as biotin functionalized magnetic nanoparticles MNP@Biotin.

8. The particle according to claim 3, characterized in that: Step (5) is to prepare SA-Tim4 functionalized magnetic nanoparticles by adding MNP@Biotin to PBS containing SVA-Tim4, pouring the mixture into a centrifuge tube, and stirring at 4°C; after the reaction is completed, using a strong magnet to isolate the nanomagnetic beads adsorbed by the heart tube wall, washing with PBS, and then storing the nanomagnetic beads in PBS, which is recorded as SA-Tim4 functionalized magnetic nanoparticles MNP-Biotin@SA-Tim4.

9. Use of the SA-Tim4 protein according to claim 1 or the SA-Tim4 functionalized magnetic nanoparticles according to any one of claims 3 to 8 in isolating exosomes.

10. A method for separating and extracting exosomes using the SA-Tim4 protein according to claim 1, characterized in that: SA-Tim4-functionalized magnetic nanoparticles were obtained by combining SA-Tim4 with biotin-modified nanomagnetic beads, and exosomes were separated by the specific binding and release of Tim4 with phosphatidylserine on the surface of exosomes.

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