Preparation method of mammalian extracellular vesicles

Abstract

The invention relates to a preparation method of mammalian extracellular vesicles. The preparation method comprises the step of separating from a recombinant mammalian cell culture solution for expressing FREE1 protein to obtain the mammalian extracellular vesicles. The FREE1 protein with the function of participating in the transportation process of the plant vesicles is expressed in mammalian cells, so that the yield of the mammalian extracellular vesicles can be remarkably increased, and the beneficial effect is not limited to common mammalian extracellular vesicles which are not loaded with goods; and for mammalian extracellular vesicles loaded with therapeutic drugs (such as interleukin and anti-inflammatory protein), the effect of remarkably increasing the yield is also achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to a method for preparing mammalian extracellular vesicles. Background Art

[0002] Extracellular vesicles are a group of vesicles with a lipid bilayer membrane structure secreted by living cells. According to their size, they can be divided into exosomes, microvesicles and apoptotic bodies. Extracellular vesicles originate from the endocytic pathway and are released to the outside of the cell by the fusion of multivesicular bodies in the cell with the plasma membrane. They can mediate the intercellular transfer of intracellular substances and can therefore be used as drug delivery carriers in vivo. Among them, exosomes, as a natural nanoparticle, have great potential as a drug delivery platform. Considering biocompatibility, immunogenicity, safety and toxicity, and the difficulty of extraction and purification, mammalian extracellular vesicles are a more recommended choice.

[0003] Extracellular vesicle drugs (drugs with extracellular vesicles as carriers) have extremely high dosage requirements, while the high cost of preparing mammalian extracellular vesicles is not conducive to their drug development. The mechanism of vesicle production in mammalian cells depends on the ESCRT / ATP pathway, that is, in the process of extracellular vesicle production in mammalian cells, ESCRT protein complexes, COP vesicle transport protein complexes, etc. need to be assembled, and a large amount of ATP is required. Due to the existence of these key rate-limiting steps, under normal circumstances, the yield of extracellular vesicles secreted by mammalian cells is limited and there is a physiological limit. Considering the extremely high loss rate and extremely low yield of extracellular vesicles during extraction and purification, the cost of mammalian extracellular vesicles is still high at the current stage, making its drug development prospects not optimistic. Therefore, how to effectively increase the yield of mammalian extracellular vesicles has always been a key bottleneck in the field of innovative extracellular vesicle drugs.

[0004] At present, some attempts have been made in this field to increase the production of extracellular vesicles, including optimization of culture conditions and optimization of extraction and purification methods. The latter has a limited effect on the increase in extracellular vesicle production. The former mainly adds some small molecules during the cell culture process, such as calcium ion carriers, ceramides, etc., to affect the growth and metabolism of cells, hoping to increase the efficiency of extracellular vesicle secretion by single cells. However, the results of these attempts are not satisfactory. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a new method for preparing mammalian extracellular vesicles which can more stably and effectively increase the yield of mammalian extracellular vesicles.

[0006] A first aspect of the present invention provides a method for preparing mammalian extracellular vesicles, comprising the step of isolating the mammalian extracellular vesicles from a culture fluid of recombinant mammalian cells expressing a FREE1 protein.

[0007] FREE1 protein is a protein involved in the plant vesicle transport process. The FREE1 protein in the present invention can be any FREE1 protein that has the function of participating in the plant vesicle transport process. FREE1 protein can come from plants or microorganisms. FREE1 protein sequences from different plant or microbial sources have comparable effects in promoting the production of extracellular vesicles in mammals. In a specific embodiment of the present invention, FREE1 protein can be a combination of one or more of Arabidopsis thaliana FREE1 protein or a FREE1 protein with a homology of more than 85%, wheat FREE1 protein, rice FREE1 protein, liverwort FREE1 protein, and beard algae FREE1 protein, etc., plant or microbial Free1 proteins.

[0008] According to some specific embodiments of the present invention, the amino acid sequence of the FREE1 protein is shown in SEQ ID NO:3.

[0009] According to the present invention, when referring to an amino acid sequence, unless otherwise specified, the amino acid sequence referred to includes not only sequences that are completely identical to the sequence but also sequences that are easily conceivable by a person skilled in the art based on the sequence and can be expected to have equivalent effects (for example, a person skilled in the art should know that sequences with high identity (for example, more than 85%, particularly more than 90%, and especially more than 95%) usually have equivalent effects).

[0010] According to a specific embodiment of the present invention, the following steps are included: (1) Construct and prepare a recombinant expression vector for expressing FREE1 protein; (2) using the recombinant expression vector to transfect mammalian cells to obtain recombinant mammalian cells; (3) culturing the recombinant mammalian cells to obtain a recombinant mammalian cell culture fluid; (4) Isolating the animal extracellular vesicles from the recombinant mammalian cell culture fluid.

[0011] According to the present invention, the mammalian cells involved include but are not limited to cells of common mammals in the art, for example, cells of cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, including but not limited to cells of non-human primates, for example, monkeys, such as cynomolgus monkeys or rhesus monkeys, chimpanzees, including but not limited to human cells. The mammalian cells involved include cells of transgenic mammals.

[0012] In an embodiment of the present invention, the host cell of the recombinant mammalian cell includes one or more of an ovarian cell line, a fibroblast cell line, an embryonic kidney cell line, a kidney cell line, a mesenchymal stem cell line, an induced stem cell line, a megakaryocyte cell line, a platelet cell line, or a lymphocyte line. Exemplary host cells include hamster ovary (CHO cell), human embryonic kidney 293 cells (e.g., HEK293, HEK293E, HEK293T), COS cells, BHK cells, NIH3T3 cells, lymphocyte lines (e.g., NS0 myeloma cells and Sp2 / 0 cells), mammary epithelial cells, induced stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, or placental mesenchymal stem cells, etc.

[0013] In the embodiment of the present invention, the expression vector used by the recombinant expression vector can be an expression vector conventionally used in the art, including a viral plasmid vector and a eukaryotic expression plasmid vector, and preferably a eukaryotic expression plasmid vector is used.

[0014] In some specific embodiments of the present invention, the expression vector used by the recombinant expression vector is a pAAVS1 plasmid, a pIRES plasmid, or a pLENTI plasmid.

[0015] In an embodiment of the present invention, the separation can adopt an extracellular vesicle separation and purification method known in the art, including one or more of ultracentrifugation separation, density gradient centrifugation separation, size exclusion chromatography separation, affinity chromatography separation, adsorption chromatography separation, bonded phase chromatography separation, immunoseparation or sieving separation.

[0016] According to some embodiments of the present invention, the recombinant animal cell culture fluid is first centrifuged at a centrifugal force of less than 1000g to collect the supernatant, and then the supernatant is centrifuged at a centrifugal force of 100000g~200000g to collect the precipitate, which is the mammalian extracellular vesicle.

[0017] Furthermore, the cell culture fluid of the recombinant cells is first centrifuged at a centrifugal force of 200g~500g, the supernatant is collected, and then the supernatant is centrifuged at a centrifugal force of 120000g~150000g to collect the precipitate, which is the extracellular vesicles loaded with cargo.

[0018] In the embodiment of the present invention, the recombinant mammalian cell culture medium is a serum-free culture medium.

[0019] In an embodiment of the present invention, the recombinant mammalian cells are cultured at 34° C. to 38° C. and 5% to 10% CO 2 to obtain the recombinant mammalian cell culture fluid.

[0020] In the embodiment of the present invention, the transfection operation is to mix the recombinant expression vector, the host cell, and the transfection reagent in a serum-free culture medium and incubate at 20° C. to 30° C.

[0021] The second aspect of the present invention provides a method for preparing mammalian extracellular vesicles loaded with pharmaceutically active molecules, the preparation method comprising the following steps: 1) obtaining a culture medium of recombinant mammalian cells, wherein the recombinant mammalian cells express not only a fusion protein based on an extracellular vesicle scaffold protein but also a FREE1 protein, wherein the fusion protein based on the extracellular vesicle scaffold protein is a fusion protein formed by a protein serving as an extracellular vesicle scaffold protein and a pharmaceutically active molecule or a linker for connecting a pharmaceutically active molecule; 2) separating the recombinant mammalian cell culture fluid; and, When the fusion protein based on the extracellular vesicle scaffold protein is a fusion protein formed by a protein serving as an extracellular vesicle scaffold protein and a linker for connecting a pharmaceutically active molecule, the preparation method further comprises: 3) After the separation step, the separated extracellular vesicles are mixed with pharmaceutically active molecules, so that the pharmaceutically active molecules are bound to the extracellular vesicles through the linker.

[0022] In an embodiment of the present invention, the step 1) includes: constructing and preparing a first recombinant expression vector for expressing FREE1 protein, and a second recombinant expression vector for expressing a fusion protein formed by an extracellular vesicle scaffold protein and a pharmaceutically active molecule or a linker; sequentially or simultaneously using the first recombinant expression vector and the second recombinant expression vector to transfect mammalian cells to obtain recombinant mammalian cells; and culturing the recombinant mammalian cells to obtain a recombinant mammalian cell culture fluid.

[0023] In an embodiment of the present invention, the FREE1 protein includes one or more of Arabidopsis thaliana FREE1 protein, wheat FREE1 protein, rice FREE1 protein, Marchantia polymorpha FREE1 protein, and Algae FREE1 protein.

[0024] In an embodiment of the present invention, the host cells of the recombinant mammalian cells include one or more of an ovarian cell line, a fibroblast cell line, an embryonic kidney cell line, a kidney cell line or a lymphocyte line.

[0025] In an embodiment of the present invention, the expression vectors used by the first recombinant expression vector and the second recombinant expression vector are respectively eukaryotic expression plasmid vectors or viral plasmid vectors.

[0026] In an embodiment of the present invention, the separation includes one or more of ultracentrifugation separation, density gradient centrifugation separation, size exclusion chromatography separation, affinity chromatography separation, adsorption chromatography separation, bonded phase chromatography separation, immunoseparation or sieving separation.

[0027] In the embodiments of the present invention, the recombinant mammalian cell culture medium is a serum-free culture medium.

[0028] In an embodiment of the present invention, the recombinant mammalian cells are cultured at 34° C. to 38° C. and 5% to 10% CO 2 to obtain the recombinant mammalian cell culture fluid.

[0029] In an embodiment of the present invention, other operating methods or operating conditions not mentioned in the method for preparing mammalian extracellular vesicles loaded with pharmaceutically active molecules are consistent with the operating methods or operating conditions in the method for preparing mammalian extracellular vesicles not loaded with pharmaceutically active molecules described above.

[0030] The pharmaceutical active molecules in the present invention include active molecules of preventive drugs, active molecules of therapeutic drugs and active molecules of diagnostic drugs.

[0031] In an embodiment of the present invention, the active molecule includes one or more of protein molecules and fragments thereof, polypeptide molecules and fragments thereof, nucleic acids and fragments thereof, or small molecule compounds.

[0032] In the present invention, the connection between the active pharmaceutical molecule and the extracellular vesicle scaffold protein or between the active pharmaceutical molecule and the linker can be achieved by using connection technologies such as gene fusion, chemical synthesis, enzyme catalysis connection, self-assembly into a complex, click chemistry, protein engineering, nanotechnology, etc.

[0033] In some embodiments of the present invention, when the pharmaceutically active molecule is a protein molecule and a fragment thereof and / or a polypeptide molecule and a fragment thereof, the pharmaceutically active molecule is directly or indirectly connected to the N-terminus and / or C-terminus of the extracellular vesicle scaffold protein through a linker. Whether a linker is needed can be determined based on the structure, function, stability, etc. of the extracellular vesicle scaffold protein and the effective substance.

[0034] In other embodiments of the present invention, when the pharmaceutically active molecule is a nucleic acid and a fragment thereof (e.g., RNA), the nucleic acid and its fragment can exist in the form of a protein-nucleic acid complex with a linker, and the linker is connected to an extracellular vesicle scaffold protein, thereby indirectly connecting the nucleic acid and its fragment to the extracellular vesicle scaffold protein, and then loading into the extracellular vesicle. For example, a fusion protein is formed by the extracellular vesicle scaffold protein and the protein linker, and at the same time, the protein linker and the nucleic acid and its fragment form a complex through non-covalent bond interaction.

[0035] For example, when sgRNA needs to be delivered, Cas9 protein can be used as a protein linker, and the Cas9 protein can be fused to the N-terminus and / or C-terminus of the extracellular vesicle scaffold protein. The sgRNA then forms an RNP complex with the Cas9 protein, thereby loading the sgRNA into the extracellular vesicle for delivery.

[0036] For another example, when siRNA needs to be delivered, AGO2 protein can be used as a protein linker, and the AGO2 protein can be fused to the N-terminus and / or C-terminus of the extracellular vesicle scaffold protein. The siRNA can form an RNP complex with the AGO2 protein, thereby loading the siRNA into the extracellular vesicle for delivery.

[0037] In some further embodiments of the present invention, the pharmaceutically active molecule is a small molecule compound, and the prepared extracellular vesicles that simultaneously express the FREE1 protein and the fusion protein formed by the extracellular vesicle scaffold protein and the linker can be directly mixed with the small molecule compound, the small molecule compound combines with the linker to form a complex, the linker is connected to the extracellular vesicle scaffold protein, thereby indirectly connecting the small molecule compound to the extracellular vesicle scaffold protein, and then loading it into the extracellular vesicle.

[0038] The binding of nucleic acids and fragments thereof and / or small molecule compounds to linkers is achieved by conventional techniques in the art. For example, the small molecule compound can be bound to the linker through affinity adsorption between the small molecule compound and the linker.

[0039] In the embodiments of the present invention, the protein molecules include but are not limited to various therapeutic proteins, immunogenic proteins or functional proteins (such as proteins used for gene editing, reporter gene proteins), enzyme proteins, etc.

[0040] In the embodiments of the present invention, the proteins used for gene editing include various proteins used in gene editing technology / engineering, including but not limited to various recombinases that can promote the recombination or rearrangement of specific sequences between DNA molecules and gene editing system-related proteins (i.e., proteins used in various gene editing systems). Known common gene editing-related proteins include but are not limited to Cre recombinase, Dre recombinase, Flp recombinase, RadA protein, RAD51 protein, RecA protein, Cas protein, transposon protein, zinc finger protein and TALE protein.

[0041] In the embodiments of the present invention, the polypeptide molecules include but are not limited to various therapeutic polypeptides, targeting polypeptides or partial polypeptide fragments of the above proteins, such as antibodies, cytokines, anti-inflammatory proteins or polypeptides, serum albumin, lectins, coagulation factors, etc.

[0042] In the embodiments of the present invention, the nucleic acid includes but is not limited to various functional nucleic acid molecules, such as DNA or mRNA molecules encoding proteins or polypeptides, RNA molecules with regulatory functions such as miRNA, antisense oligonucleotides ASO or siRNA, sgRNA of CRISPR system, and even plasmid DNA.

[0043] In an embodiment of the present invention, the pharmaceutically active molecule is a cytokine receptor protein, a protein for gene editing, a cytokine or RNA.

[0044] In some embodiments of the present invention, the drug active molecule is Cre recombinase, Dre recombinase, Flp recombinase, Cas protein, cGAS, srIκB, IL4, IL5, IL10, TGF-β, STAT6, A20, SIRPα, IL-1Ra, SGK1, KDM5B, 4-1BBL, TMPRSS2, CD20, CD133, CCR4, CCR7, CCR8, CXCR4, CXCR5, GIPR, GPRC5D, NaPi2b, sgRNA or siRNA.

[0045] In an embodiment of the present invention, the extracellular vesicle scaffold protein is any scaffold protein capable of enriching and loading cargo in extracellular vesicles, including but not limited to scaffold proteins known in the art. For example, the extracellular vesicle scaffold protein includes one or more of BASP1 protein or fragments thereof, PTGFRN protein or fragments thereof, TSPAN2 protein or fragments thereof, TSPAN3 protein or fragments thereof, CD63 protein or fragments thereof, CD9 protein or fragments thereof, CD81 protein or fragments thereof, LEAP protein or fragments thereof, and PVR protein or fragments thereof.

[0046] In an embodiment of the present invention, the first recombinant expression vector is used to introduce an exogenous nucleic acid fragment encoding a FREE1 protein into a mammalian cell for expression. The second recombinant expression vector is used to introduce an exogenous nucleic acid fragment encoding an "extracellular vesicle scaffold protein" or a fusion protein of a "extracellular vesicle scaffold protein-linker" or a fusion protein of a "extracellular vesicle scaffold protein-drug active molecule" into a mammalian cell for expression, thereby obtaining a recombinant mammalian cell that simultaneously overexpresses a FREE1 protein and an extracellular vesicle scaffold protein, or a recombinant mammalian cell that simultaneously overexpresses a FREE1 protein and a fusion protein formed by an extracellular vesicle scaffold protein and the linker, or a recombinant mammalian cell that simultaneously overexpresses a FREE1 protein and a fusion protein formed by an extracellular vesicle scaffold protein and a drug active molecule.

[0047] The third aspect of the present invention also provides mammalian extracellular vesicles prepared by the above preparation method.

[0048] In an embodiment of the present invention, the mammalian extracellular vesicles are exosomes, microvesicles or apoptotic bodies.

[0049] A fourth aspect of the present invention also provides mammalian extracellular vesicles loaded with pharmaceutically active molecules prepared by the above-mentioned preparation method, wherein the mammalian extracellular vesicles are exosomes, microvesicles or apoptotic bodies.

[0050] The fifth aspect of the present invention also provides the use of the above-mentioned mammalian extracellular vesicles or the above-mentioned mammalian extracellular vesicles loaded with pharmaceutically active molecules in the preparation of a pharmaceutical composition.

[0051] The sixth aspect of the present invention also provides a recombinant mammalian cell for preparing mammalian extracellular vesicles, which is the same as any of the recombinant mammalian cells mentioned above, including recombinant mammalian cells expressing FREE1 protein, recombinant mammalian cells simultaneously expressing FREE1 protein and a fusion protein formed by an extracellular vesicle scaffold protein and a pharmaceutically active molecule, and recombinant mammalian cells simultaneously expressing FREE1 protein and a fusion protein formed by an extracellular vesicle scaffold protein and a linker.

[0052] The seventh aspect of the present invention also provides a recombinant expression vector system for preparing mammalian extracellular vesicles, which includes one or both of the first recombinant expression vector and the second recombinant expression vector described above.

[0053] The eighth aspect of the present invention also provides a pharmaceutical composition, which comprises any of the mammalian extracellular vesicles mentioned above and may optionally include a pharmaceutically acceptable excipient.

[0054] In the embodiments of the present invention, specific excipients can be selected according to the intended mode of administration and therapeutic application. The pharmaceutically acceptable excipients include but are not limited to pharmaceutically, nutritionally or physiologically acceptable carriers.

[0055] Furthermore, the pharmaceutical composition includes, but is not limited to, tablets, granules, pills, capsules, emulsions, pastes, gels, suspensions, solutions, powders, transdermal patches, sprays, suppositories or implants.

[0056] A ninth aspect of the present invention provides a kit, which comprises the above-mentioned mammalian extracellular vesicles, or the above-mentioned pharmaceutical composition.

[0057] A tenth aspect of the present invention provides a method for diagnosing, preventing or treating a disease, comprising administering the above-mentioned mammalian extracellular vesicles (preferably mammalian extracellular vesicles loaded with pharmaceutically active molecules) or the above-mentioned pharmaceutical composition to a subject.

[0058] In an embodiment of the present invention, it is preferred to determine the specific disease that the mammalian extracellular vesicles correspond to for detection, prevention or treatment based on the pharmaceutically active molecules loaded into the mammalian extracellular vesicles, including but not limited to various tumor diseases or infectious diseases.

[0059] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art: The present invention can significantly increase the yield of mammalian extracellular vesicles by overexpressing plant-derived FREE1 protein in mammalian cells compared to existing methods. This beneficial effect is not limited to ordinary mammalian extracellular vesicles that are not loaded with cargo, but also has a significant effect of increasing the yield of mammalian extracellular vesicles loaded with therapeutic drugs (such as interleukins, anti-inflammatory proteins). Moreover, compared with the existing method of adding exogenous small molecule compounds to the culture medium to affect the growth and metabolism of mammalian cells, the preparation method of the present invention can stably increase the yield of mammalian extracellular vesicles while avoiding the introduction of exogenous substances into the cell culture medium, thereby reducing impurities in the cell culture fluid, so that the purity of the extracted extracellular vesicles is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the particle size distribution diagram of the exosomes in Example 1; Figure 2 This is the particle size distribution diagram of exosomes in comparative example 1; Figure 3 This is the particle size distribution diagram of exosomes in comparative example 2; Figure 4 This is the particle size distribution diagram of exosomes in comparative example 3; Figure 5 This is the particle size distribution diagram of the exosomes in Example 2; Figure 6 This is the particle size distribution diagram of exosomes in Comparative Example 4; Figure 7 This is the particle size distribution diagram of the exosomes in Example 3; Figure 8 This is the particle size distribution diagram of exosomes in comparative example 5; Fig. 9 This is a transmission electron microscopy image of the exosomes in Example 1; Fig.10 This is a transmission electron microscopy image of the exosomes in Comparative Example 1; Fig.11 This is a transmission electron microscopy image of the exosomes in comparative example 2; Fig.12 This is a transmission electron microscopy image of the exosomes of Comparative Example 3; Fig.13 This is a comparison chart of the yield of exosomes loaded with IL15 interleukin in the supernatant of Example 2 and Comparative Example 4; Fig.14 This is a comparison chart of the yield of exosomes loaded with srIkB anti-inflammatory protein in the supernatant of Example 3 and Comparative Example 5. DETAILED DESCRIPTION

[0061] The mechanism of mammalian cells producing extracellular vesicles depends on only one pathway, ESCRT / ATP, and the production of extracellular vesicles secreted by this pathway is very limited. FREE1 protein is a protein involved in the plant vesicle transport process and is only expressed in plant cells. The present invention overexpresses plant-derived FREE1 protein in mammalian cells and unexpectedly finds that it can effectively promote mammalian cells to produce more extracellular vesicles, and also has the effect of promoting the stable increase of the production of exosomes loaded with cargo.

[0062] The technical solution of the present invention is further illustrated below in conjunction with specific embodiments and comparative examples.

[0063] In the present invention, it should be noted that, unless otherwise specified or contradictory to the context, the terms or expressions used in this article should be read in conjunction with the overall content of this article and as understood by those of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art.

[0064] "FREE1 protein" is a protein involved in the plant vesicle transport process. The FREE1 protein in the present invention includes any FREE1 protein in the art that has the function of participating in the plant vesicle transport process, including FREE1 proteins from different plant sources.

[0065] "Extracellular vesicles" are a group of vesicles with a lipid bilayer membrane structure secreted by living cells. According to their size, they can be divided into: exosomes, microvesicles and apoptotic bodies.

[0066] "Exosomes" refer to cell-derived extracellular vesicles (EVs) with a diameter of 30~150nm, which include a membrane that encloses the internal space. The membrane is a phospholipid bilayer structure. Exosomes can mediate the intercellular transfer of intracellular substances.

[0067] “Loading” refers to the attachment and enrichment of cargo into extracellular vesicles, including various forms of cargo in the lumen of extracellular vesicles, on the membrane of extracellular vesicles, or attached to the outside of the membrane of extracellular vesicles.

[0068] "Extracellular vesicle scaffold protein" refers to a linker used to connect a cargo to an extracellular vesicle, which is used to anchor any cargo of interest to an extracellular vesicle.

[0069] "Cargo" mainly refers to "pharmaceutically active molecules", i.e., proteins and fragments thereof, polypeptides and fragments thereof, nucleic acids and fragments thereof, or small molecule compounds of any functional interest (e.g., preventive, therapeutic, diagnostic, etc.). "Cargo" is also often referred to as "functional cargo" or "effective substance".

[0070] "Small molecule compound" refers to a compound with a molecular size less than 1000 molecular weight, including conventional organic compounds and inorganic compounds.

[0071] "Polypeptide" and "oligopeptide" are defined according to the length of the peptide chain. Those with a length of 10 to 100 amino acids are called "polypeptides", and those with a length of less than 10 amino acids are called "oligopeptides".

[0072] "Recombinase" is a class of enzymes that can promote the recombination or rearrangement of modified sequences between DNA molecules. Common recombinases can be loaded and delivered using the method of the present invention. Common recombinases include but are not limited to Cre recombinase, Dre recombinase, Flp recombinase, RadA protein, RAD51 protein and RecA protein.

[0073] "Cytokines" are a class of small molecule proteins with a wide range of biological activities that are synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. Cytokines generally regulate cell growth, differentiation and effects by binding to corresponding receptors, and regulate immune responses. They have multiple functions such as regulating innate immunity and adaptive immunity, hematopoiesis, cell growth, and damaged tissue repair. Common cytokines include, but are not limited to, pro-inflammatory cytokines, anti-inflammatory cytokines, interleukins, interferons, growth factors, and chemokines.

[0074] "Gene editing proteins" are a class of proteins used in gene editing systems. Common gene editing proteins include Cas proteins (such as Cas9, Cas12, Cas13, Cas2, Cas3, Cas10, Cas14, Cas12f, CasX, CasY or Csm), transposon proteins, zinc finger proteins and TALE proteins.

[0075] "Exogenous nucleic acid fragments" include any gene or fragment thereof of interest, such as a gene or fragment thereof encoding a scaffold protein. Exogenous nucleic acid fragments have a different origin from the host cell, for example, nucleic acid sequences isolated from an organism different from the host cell, i.e., are exogenous nucleic acid fragments relative to the host cell.

[0076] "Plasmid" refers to a circular double-stranded DNA that is capable of accepting foreign nucleic acid fragments and replicating in prokaryotic or eukaryotic cells.

[0077] "Promoter" refers to a nucleic acid sequence that can control the transcription of a coding sequence. The promoter sequence includes a modified sequence that is sufficient to allow RNA polymerase to recognize, bind and initiate transcription. A promoter can affect the transcription of a gene located on the same nucleic acid molecule as itself or a gene located on a different nucleic acid molecule from itself.

[0078] "Host cells" are also called recipient cells, and exemplary host cells include human embryonic kidney cells HEK293. It is understood that the progeny of a single parental cell may not necessarily be completely identical to the original parent in morphology or in genomic or total DNA complement due to natural, accidental, or deliberate mutation.

[0079] The technical solution of the present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0080] The implementation conditions used in the following examples and comparative examples can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments. The instruments, raw materials and reagents not specifically specified can be obtained commercially.

[0081] Example 1 This embodiment provides a method for preparing exosomes, which is as follows: 1. Construction of Plasmid A recombinant expression vector for expressing FREE1 protein was constructed using pIRES plasmid as an expression vector. The expression cassette of the first recombinant expression vector was designed as follows: using CMV promoter (sequence as shown in SEQ ID NO: 1) as the transcription start point, using SV40 polyA (sequence as shown in SEQ ID NO: 2) as the transcription end point, and expressing FREE1 protein (sequence as shown in SEQ ID NO: 3).

[0082] The recombinant expression vector was prepared according to the following specific steps: (1) PCR cloning: Use conventional methods in the art to design upstream primers and downstream primers according to the expression frame, and synthesize upstream primers, downstream primers and template DNA. In a 0.2mL EP tube, add 25μL DNA polymerase (2×Phanta MaxMaster Mix, Vazyme, P515-01), 2μL upstream primer, 2μL downstream primer, 10g template DNA, and add enzyme-free water (RNase-free dd H2O, Vazyme, P071-01-AA) to 50μL, mix well, and put into the PCR instrument, and design the amplification program according to the primer annealing temperature.

[0083] (2) Purification of PCR products: Purify the PCR products according to the kit instructions (Vazyme, DC301-01).

[0084] (3) Enzyme digestion: Add 5 μL rCutSmart TM Buffer (BioLabs, 136004s), 0.5μL Pac I enzyme (BioLabs, R0547L), 0.5μL Not I enzyme (BioLabs, R3189L), 2μg PCR purified fragment, 5μg pAAVS1 vector plasmid. Add enzyme-free water (RNase-free ddH2O, Vazyme, P071-01-AA) to 10μL, mix well, and place in a PCR instrument for enzyme digestion at 37℃ for 16h.

[0085] (4) Ligation transformation: In a 0.2 mL EP tube, add 4 μL of the digestion solution obtained in step (3), 1 μL of T4 DNA ligase (Vazyme, N103-01-AA) and 1 μL of T4 DNA ligase buffer (Vazyme, N103-01-AC). After incubating at room temperature for 10 min, take out 5 μL and add it to competent cells (DH5α, Vazyme, C502-03), incubate on ice for 30 min, incubate at 42°C for 90 s, incubate on ice for 2 min, then add 1 mL LB medium (BeyoPure™, 04.05.ST156) and culture in a shaking incubator for 1 h. After culture, centrifuge at 12000g for 1 min, remove the supernatant and keep 100 μL, resuspend and add to the bacterial culture dish (White Shark Biosharp, BS-90-D), then use a disposable plastic coating rod (White Shark Biosharp, BS-PS-A) to spread evenly and store in a 37°C constant temperature incubator for later use.

[0086] Wherein, in step (1), the upstream primer sequence designed according to the expression frame of the recombinant expression vector for expressing FREE1 protein is shown in SEQ ID NO:4, the downstream primer sequence is shown in SEQ ID NO:5, and the template DNA sequence is shown in SEQ ID NO:6.

[0087] 2. Cell Transfection The specific steps are as follows: (1) Prepare cells for transfection EXPI293F cells were inoculated in a 125 mL shake flask at 1.5E+6 / mL with a survival rate of more than 95%. The total volume of serum-free cell culture medium (Optima medium, CD05) was 25 mL. The cells were placed in an 8% CO2 constant temperature shaker at 37°C and 100 rpm for overnight culture, and transfection was started the next day.

[0088] (2) PEI transfection The transfection process using TA293 reagent (Zhuhai Kairui Biotechnology Co., Ltd., K2001) is as follows: The target plasmid is extracted from the transformed competent cells preserved by the above-mentioned coating culture by conventional technical means in the art, and the recombinant expression vector constructed for expressing FREE1 protein is obtained. Prepare two 15mL centrifuge tubes, add 1.25mL serum-free cell culture medium and 25μg target plasmid to one of them, and vortex to mix; add 1.25mL serum-free cell culture medium and 125μL TA293 transfection reagent to the other, and vortex to mix. Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the target plasmid and vortex to mix. Incubate at room temperature for 10 minutes to prepare the plasmid-vector complex. Take out the EXPI293F cells from the constant temperature shaker, add the prepared plasmid-vector complex dropwise while shaking, and then put it back into the 8% CO2 constant temperature shaker, and culture it at 37°C and 100rpm for 72 hours.

[0089] 3. Isolation and purification of exosomes After 72 hours of shaking culture, the cell culture fluid was first centrifuged at 300g, 4°C, for 5 minutes, and the supernatant was collected. The supernatant was then ultracentrifuged at 133900g, 4°C for 1 hour to collect the precipitate. The precipitate (i.e., the isolated exosomes) was resuspended in 100 μL PBS and stored at 4°C for later use.

[0090] Comparative Example 1 This comparative example directly uses EXPI293F cells to prepare exosomes, as follows: 1. Inoculate EXPI293F cells at 1.5E+6 / mL with a survival rate of more than 95% in a 125mL shake flask with a total volume of 25mL of serum-free cell culture medium (Optima medium, CD05). Place in an 8% CO2 constant temperature shaker at 37°C and 100rpm for 72 hours.

[0091] 2. After 72 hours of shaking culture, the cell culture fluid was first centrifuged at 300g, 4℃, low speed for 5min, and the supernatant was collected. Then the supernatant was ultracentrifuged at 133900g, 4℃ for 1h to collect the precipitate. The precipitate (i.e., the isolated exosomes) was resuspended in 100μL PBS and stored at 4℃ for later use.

[0092] Comparative Example 2 This comparative example provides another method for preparing exosomes. In this comparative example, a calcium ion carrier (Calcimycin A23187) with a final concentration of 10 μM is added to the serum-free culture medium, as follows: 1. Inoculate EXPI293F cells at 1.5E+6 / mL with a survival rate of more than 95% in a 125mL shake flask. The total volume of serum-free cell culture medium (Optima medium, CD05) is 25mL. Calicimycin with a final concentration of 10μM is added to the serum-free cell culture medium. Place in an 8% CO2 constant temperature shaker at 37°C and 100rpm for 72 hours.

[0093] 2. After 72 hours of shaking culture, the cell culture fluid was first centrifuged at 300g, 4℃, low speed for 5min, and the supernatant was collected. Then the supernatant was ultracentrifuged at 133900g, 4℃ for 1h to collect the precipitate. The precipitate (i.e., the isolated exosomes) was resuspended in 100μL PBS and stored at 4℃ for later use.

[0094] Comparative Example 3 This comparative example provides another method for preparing exosomes. In this comparative example, ceramide with a final concentration of 100 μg / mL is added to the serum-free medium, as follows: 1. Inoculate EXPI293F cells at 1.5E+6 / mL with a survival rate of more than 95% in a 125mL shake flask. The total volume of serum-free cell culture medium (Optima medium, CD05) is 25mL. Serum-free cell culture medium is supplemented with ceramide at a final concentration of 100μg / mL. Place in an 8% CO2 constant temperature shaker at 37°C and 100rpm for 72 hours.

[0095] 2. After 72 hours of shaking culture, the cell culture fluid was first centrifuged at 300g, 4℃, low speed for 5min, and the supernatant was collected. Then the supernatant was ultracentrifuged at 133900g, 4℃ for 1h to collect the precipitate. The precipitate (i.e., the isolated exosomes) was resuspended in 100μL PBS and stored at 4℃ for later use.

[0096] Example 2 This embodiment provides a method for preparing exosomes loaded with IL15 interleukin, which is as follows: 1. Construction of recombinant expression plasmid vector system According to the method of Example 1, a recombinant expression vector for expressing FREE1 protein was constructed and prepared.

[0097] Using pIRES plasmid as an expression vector and PTGFRN protein (sequence as shown in SEQ ID NO:7) as an exosome scaffold protein, a recombinant expression vector for expressing a cargo (IL15 interleukin, sequence as shown in SEQ ID NO:8) was constructed, and the expression cassette was designed as follows: using CMV promoter (sequence as shown in SEQ ID NO:1) as a transcription start point, using SV40 polyA (sequence as shown in SEQ ID NO:2) as a transcription end point, expressing a fusion protein of IL15 interleukin and PTGFRN, and IL15 interleukin was connected to the N-terminus of the PTGFRN protein. A recombinant expression vector for expressing a fusion protein of IL15 interleukin and PTGFRN was prepared according to the method of Example 1, wherein the upstream primer sequence designed according to the expression cassette of the recombinant expression vector for expressing a fusion protein of IL15 interleukin and PTGFRN is shown in SEQ ID NO:9, the downstream primer sequence is shown in SEQ ID NO:10, and the template DNA sequence is shown in SEQ ID NO:11.

[0098] 2. Cell Transfection The specific steps are as follows: (1) Prepare cells for transfection EXPI293F cells were inoculated in a 125 mL shake flask at 1.5E+6 / mL with a survival rate of more than 95%. The total volume of serum-free cell culture medium (Optima medium, CD05) was 25 mL. The cells were placed in an 8% CO2 constant temperature shaker at 37°C and 100 rpm for overnight culture, and transfection was started the next day.

[0099] (2) PEI transfection The transfection process using TA293 reagent (Zhuhai Kairui Biotechnology Co., Ltd., K2001) is as follows: The target plasmid is extracted from the transformed competent cells preserved by the above-mentioned coating culture by conventional technical means in the art, that is, the constructed recombinant expression vector for expressing FREE1 protein and the recombinant expression vector for expressing the fusion protein of IL15 interleukin and PTGFRN are obtained. Prepare two 15mL centrifuge tubes, add 1.25mL serum-free cell culture medium, 25μg target plasmid (a mixture of the recombinant expression vector for expressing FREE1 protein and the recombinant expression vector for expressing the fusion protein of IL15 interleukin and PTGFRN in a mass ratio of 1:1) to one of them, and vortex mix; add 1.25mL serum-free cell culture medium and 125μL TA293 transfection reagent to the other, and vortex mix. Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the target plasmid and vortex mix. Incubate at room temperature for 10 minutes to prepare a plasmid-vector complex. The EXPI293F cells were removed from the thermostatic shaker, and the prepared plasmid-vector complex was added dropwise while shaking. The cells were then placed back into the 8% CO2 thermostatic shaker and cultured at 37°C and 100 rpm for 72 hours.

[0100] 3. Isolation and purification of exosomes After 72 hours of shaking culture, the cell culture fluid was first centrifuged at 300g, 4°C, for 5 minutes, and the supernatant was collected. The supernatant was then ultracentrifuged at 133900g, 4°C for 1 hour to collect the precipitate. The precipitate (i.e., the isolated exosomes loaded with IL15 interleukin) was resuspended in 100μL PBS and stored at 4°C for later use.

[0101] Comparative Example 4 This comparative example provides another method for preparing exosomes loaded with IL15 interleukin. The host cells (EXPI293F cells) of this comparative example do not express FREE1 protein. The specific steps of this comparative example are as follows: 1. Inoculate EXPI293F cells at 1.5E+6 / mL with a survival rate of more than 95% in a 125mL shake flask with a total volume of 25mL of serum-free cell culture medium (Optima medium, CD05). Place in an 8% CO2 constant temperature shaker at 37°C and 100rpm overnight, and start transfection the next day.

[0102] 2. Prepare two 15mL centrifuge tubes, add 1.25mL serum-free cell culture medium and 25μg target plasmid (the recombinant expression vector for expressing the fusion protein of IL15 interleukin and PTGFRN prepared in Example 2) to one of them, and vortex to mix; add 1.25mL serum-free cell culture medium and 125μL TA293 transfection reagent to the other, and vortex to mix. Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the target plasmid and vortex to mix. Incubate at room temperature for 10 minutes to prepare the plasmid-vector complex. Remove the EXPI293F cells from the constant temperature shaker, add the prepared plasmid-vector complex dropwise while shaking, and then return to the 8% CO2 constant temperature shaker, 37°C, 100rpm shaking culture for 72 hours.

[0103] 3. After 72 hours of shaking culture, the cell culture fluid was first centrifuged at 300g, 4℃, for 5 minutes, and the supernatant was collected. Then the supernatant was ultracentrifuged at 133900g, 4℃ for 1 hour to collect the precipitate. The precipitate (i.e., the separated exosomes loaded with IL15 interleukin) was resuspended in 100μL PBS and stored at 4℃ for later use.

[0104] Example 3 This embodiment provides an exosome loaded with srIkB anti-inflammatory protein.

[0105] In this embodiment, a recombinant expression vector for expressing a cargo (srIkB anti-inflammatory protein, sequence shown in SEQ ID NO: 13) was constructed using a pIRES plasmid as an expression vector and Basp1 protein (sequence shown in SEQ ID NO: 12) as an exosome scaffold protein. The expression cassette was designed as follows: a CMV promoter (sequence shown in SEQ ID NO: 1) was used as a transcription start point, and a SV40 polyA (sequence shown in SEQ ID NO: 2) was used as a transcription end point to express a fusion protein of srIkB anti-inflammatory protein and Basp1 protein, and srIkB anti-inflammatory protein was connected to the C-terminus of Basp1 protein. A recombinant expression vector for expressing a fusion protein of srIkB anti-inflammatory protein and Basp1 protein was prepared according to the method of Example 1, wherein the upstream primer sequence designed according to the expression cassette of the recombinant expression vector for expressing a fusion protein of srIkB anti-inflammatory protein and Basp1 protein is shown in SEQ ID NO: 14, the downstream primer sequence is shown in SEQ ID NO: 15, and the template DNA sequence is shown in SEQ ID NO: 16.

[0106] According to the method of Example 2, EXPI293F cells were transfected with a mixture of a recombinant expression vector for expressing FREE1 protein and a recombinant expression vector for expressing srIkB anti-inflammatory protein and Basp1 protein in a mass ratio of 1:1, and then cultured and isolated exosomes loaded with srIkB anti-inflammatory protein.

[0107] Comparative Example 5 This comparative example provides another method for preparing exosomes loaded with srIkB anti-inflammatory protein. The host cells (EXPI293F cells) of this comparative example do not express FREE1 protein. Except that the recombinant expression vector for expressing the fusion protein of IL15 interleukin and PTGFRN in comparative example 4 is replaced with the recombinant expression vector for expressing srIkB anti-inflammatory protein and Basp1 protein prepared in Example 3, the other operations are the same as comparative example 4.

[0108] Example 4 This embodiment provides a method for preparing exosomes loaded with sgRNA, and the sequence of sgRNA is shown in SEQ ID NO: 17. In this embodiment, referring to the method in Example 3, Basp1 protein is used as the exosome scaffold protein to prepare exosomes loaded with Cas9 protein (sequence shown in SEQ ID NO: 18), and then sgRNA is mixed with exosomes loaded with Cas9 protein, and Cas9 protein is used as a protein linker. The sgRNA and Cas9 protein form an RNP complex, thereby loading the sgRNA onto the exosomes to obtain exosomes loaded with sgRNA.

[0109] Example 5 This embodiment provides a method for preparing exosomes loaded with a small molecule compound (rapamycin), using FKBP12 protein (sequence as shown in SEQ ID NO: 19) as a protein linker. In this embodiment, referring to the method of Example 3, Basp1 protein is used as an exosome scaffold protein to prepare exosomes loaded with FKBP12 protein, and then rapamycin is mixed with the exosomes loaded with FKBP12 protein, and rapamycin and FKBP12 protein form a complex, thereby loading rapamycin onto the exosomes to obtain exosomes loaded with small molecule compounds.

[0110] (1) The exosome resuspensions of the above examples and comparative examples were subjected to NTA analysis using a ZetaView instrument (Fuliu Bio Particle Metrix, Catalog No. N30E). The particle size distribution of the exosomes in Example 1 is shown in Figure 1 The particle size distribution of exosomes in comparative example 1 is shown in Figure 2 The particle size distribution of exosomes in comparative example 2 is shown in Figure 3 The particle size distribution of exosomes in comparative example 3 is shown in Figure 4The particle size distribution of the exosomes loaded with IL15 interleukin in Example 2 is shown in Figure 5 The particle size distribution of the exosomes loaded with IL15 interleukin in comparative example 4 is shown in Figure 6 The particle size distribution of the exosomes loaded with srIkB anti-inflammatory protein in Example 3 is shown in Figure 7 The particle size distribution of the exosomes loaded with srIkB anti-inflammatory protein in comparative example 5 is shown in Figure 8 The average particle size and median particle size are shown in Table 1.

[0111]

[0112] Table 1 shows that the particle sizes of the exosomes in the examples and comparative examples are all between 110 and 170 nm. Among them, IL15 interleukin is loaded on the membrane surface of the exosomes, and srIkB anti-inflammatory protein is loaded in the cavity of the exosomes. Therefore, the particle size of the exosomes loaded with IL15 interleukin in Example 2 is larger than that of the exosomes loaded with srIkB anti-inflammatory protein in Example 3.

[0113] (2) The structures of the exosomes prepared in Example 1 and Comparative Examples 1 to 3 were observed under a transmission electron microscope (TEM). Figure 9~Figure 12 As shown, the exosomes of Example 1 and Comparative Examples 1 to 3 all showed typical exosome vesicle structures (cup-shaped or saucer-shaped) under an electron microscope, and the exosomes of Example 1 had a cleaner background and higher purity.

[0114] (3) Comparison of the yield of cargo-loaded exosomes Take equal volumes of the supernatant from Example 2 and Comparative Example 4 for WB analysis. The electrophoresis results are as follows: Fig.13 As shown, the band of IL15 interleukin in Example 2 is brighter, indicating that the yield of exosomes loaded with IL15 interleukin in the supernatant of Example 2 is higher than that of Comparative Example 4.

[0115] Take equal volumes of the supernatant from Example 3 and Comparative Example 5 for WB analysis. The electrophoresis results are as follows: Fig.14 As shown, the band of the srIkB anti-inflammatory protein in Example 3 is brighter, indicating that the yield of exosomes loaded with srIkB anti-inflammatory protein in the supernatant of Example 3 is higher than that in Comparative Example 5.

[0116] (4) The above-mentioned embodiments and comparative examples were repeated three times in batches, and the exosome particle number concentration of the three batches of exosome heavy suspension was analyzed by NTA. The exosome yield per unit volume of cell supernatant and the exosome yield per single cell were back-calculated. The average exosome yield per unit volume of cell supernatant and the average exosome yield per single cell of the three batches of each embodiment and each comparative example are shown in Table 2.

[0117]

[0118] The calculation method of exosome yield per unit volume of cell supernatant was as follows: exosome particle number concentration obtained by NTA analysis × total volume of exosomes after PBS resuspension ÷ total volume of cell supernatant collected by low-speed centrifugation.

[0119] The calculation method for the exosome yield of a single cell is: the number of exosome particles obtained by NTA analysis × the total volume of exosomes after PBS resuspension ÷ the total number of cells in the cell culture medium when the exosomes were harvested. The total number of cells was detected using a cell counter and trypan blue staining method.

[0120] Table 2 shows that compared with Comparative Example 1, Comparative Example 2 and Comparative Example 3 did not increase the exosome yield, while Example 1 can significantly increase the exosome yield, with the exosome yield per mL of cell supernatant increased by 91%, and the exosome yield per cell increased by 142%. Compared with Comparative Example 4, Example 2 can increase the exosome yield per mL of cell supernatant of exosomes loaded with IL15 interleukin by 33%, and the exosome yield per cell by 68%. Compared with Comparative Example 5, Example 3 can increase the exosome yield per mL of cell supernatant of exosomes loaded with srIkB anti-inflammatory protein by 118%, and the exosome yield per cell by 187%.

[0121] The above results show that the present invention can significantly increase the yield of exosomes by overexpressing plant-derived FREE1 protein in mammalian cells. This beneficial effect is not limited to ordinary exosomes, but also has a significant effect of increasing the yield of exosomes loaded with therapeutic drugs such as interleukins, anti-inflammatory proteins, etc. In addition, the background of exosomes extracted by the method of the present invention is cleaner under the electron microscope, indicating that the purity is higher.

[0122] The above detailed description of the present invention is intended to enable those skilled in the art to understand the contents of the present invention and implement them, but it does not limit the protection scope of the present invention. The present invention is not limited to the above embodiments, and any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing mammalian extracellular vesicles, characterized in that: The method comprises the step of isolating the mammalian extracellular vesicles from the culture fluid of recombinant mammalian cells expressing FREE1 protein.

2. The preparation method according to claim 1, characterized in that: The FREE1 protein includes one or more of Arabidopsis thaliana FREE1 protein, wheat FREE1 protein, rice FREE1 protein, Marchantia polymorpha FREE1 protein, and Algae FREE1 protein.

3. The preparation method according to claim 1, characterized in that: The following steps are involved: (1) Construct and prepare a recombinant expression vector for expressing FREE1 protein; (2) using the recombinant expression vector to transfect mammalian cells to obtain recombinant mammalian cells; (3) culturing the recombinant mammalian cells to obtain a recombinant mammalian cell culture fluid; (4) Isolating the animal extracellular vesicles from the recombinant mammalian cell culture fluid.

4. The preparation method according to claim 3, characterized in that: The host cell of the recombinant mammalian cell includes one or more of an ovarian cell line, a fibroblast cell line, an embryonic kidney cell line, a kidney cell line, a mesenchymal stem cell line, an induced stem cell line, a megakaryocyte cell line, a platelet cell line or a lymphocyte cell line; And / or, the expression vector used by the recombinant expression vector is a eukaryotic expression plasmid vector or a viral plasmid vector; and / or, the separation comprises one or more of ultracentrifugation separation, density gradient centrifugation separation, size exclusion chromatography separation, affinity chromatography separation, adsorption chromatography separation, bonded phase chromatography separation, immunoseparation or sieving separation; And / or, the recombinant mammalian cell culture medium is a serum-free medium; And / or, the recombinant mammalian cells are cultured at 34° C. to 38° C. and 5% to 10% CO 2 to obtain the recombinant mammalian cell culture fluid.

5. The preparation method according to claim 3, characterized in that: The host cell of the recombinant mammalian cell is one or more of CHO cells, HEK293 cells, BHK cells, COS cells, NIH3T3 cells, mammary epithelial cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, induced stem cells, NS0 cells or Sp2 / 0 cells; And / or, the expression vectors used by the recombinant expression vector are independently pAAVS1 plasmid, pIRES plasmid or pLENTI plasmid.

6. A method for preparing mammalian extracellular vesicles loaded with pharmaceutically active molecules, characterized in that: The preparation method comprises the following steps: 1) obtaining a culture medium of recombinant mammalian cells, wherein the recombinant mammalian cells express not only a fusion protein based on an extracellular vesicle scaffold protein but also a FREE1 protein, wherein the fusion protein based on the extracellular vesicle scaffold protein is a fusion protein formed by a protein serving as an extracellular vesicle scaffold protein and a pharmaceutically active molecule or a linker for connecting a pharmaceutically active molecule; 2) separating the recombinant mammalian cell culture fluid; and, When the fusion protein based on the extracellular vesicle scaffold protein is a fusion protein formed by a protein serving as an extracellular vesicle scaffold protein and a linker for connecting a pharmaceutically active molecule, the preparation method further comprises: 3) After the separation step, the separated extracellular vesicles are mixed with pharmaceutically active molecules, so that the pharmaceutically active molecules are bound to the extracellular vesicles through the linker.

7. The preparation method according to claim 6, characterized in that: The step 1) includes: constructing and preparing a first recombinant expression vector for expressing FREE1 protein, and a second recombinant expression vector for expressing a fusion protein formed by an extracellular vesicle scaffold protein and a pharmaceutically active molecule or a linker; sequentially or simultaneously using the first recombinant expression vector and the second recombinant expression vector to transfect mammalian cells to obtain recombinant mammalian cells; and culturing the recombinant mammalian cells to obtain a recombinant mammalian cell culture fluid.

8. The preparation method according to claim 7, characterized in that: The FREE1 proteins include one or more of Arabidopsis thaliana FREE1 protein, wheat FREE1 protein, rice FREE1 protein, Marchantia polymorpha FREE1 protein, and Algae FREE1 protein; and / or, the host cells of the recombinant mammalian cells include one or more of an ovarian cell line, a fibroblast cell line, an embryonic kidney cell line, a kidney cell line or a lymphocyte cell line; And / or, the expression vectors used by the first recombinant expression vector and the second recombinant expression vector are respectively eukaryotic expression plasmid vectors or viral plasmid vectors; and / or, the separation comprises one or more of ultracentrifugation separation, density gradient centrifugation separation, size exclusion chromatography separation, affinity chromatography separation, adsorption chromatography separation, bonded phase chromatography separation, immunoseparation or sieving separation; And / or, the culture adopts serum-free medium; And / or, the culturing is carried out at 34°C to 38°C and 5% to 10% CO2; And / or, the pharmaceutically active molecule includes one or more of protein molecules and fragments thereof, polypeptide molecules and fragments thereof, nucleic acids and fragments thereof, or small molecule compounds; And / or, when the pharmaceutically active molecule is a protein molecule and a fragment thereof and / or a polypeptide molecule and a fragment thereof, the fusion protein based on the extracellular vesicle scaffold protein is a fusion protein formed by a protein serving as an extracellular vesicle scaffold protein and the pharmaceutically active molecule; And / or, when the pharmaceutically active molecule is a nucleic acid and a fragment thereof and / or a small molecule compound, the fusion protein based on the extracellular vesicle scaffold protein is a fusion protein formed by a protein serving as the extracellular vesicle scaffold protein and a linker; And / or, the extracellular vesicle scaffold protein includes one or more of BASP1 protein or a fragment thereof, PTGFRN protein or a fragment thereof, TSPAN2 protein or a fragment thereof, TSPAN3 protein or a fragment thereof, CD63 protein or a fragment thereof, CD9 protein or a fragment thereof, CD81 protein or a fragment thereof, LEAP protein or a fragment thereof, and PVR protein or a fragment thereof.

9. A mammalian extracellular vesicle, characterized in that The mammalian extracellular vesicles are exosomes, microvesicles or apoptotic bodies prepared by the preparation method according to any one of claims 1 to 5.

10. A mammalian extracellular vesicle loaded with a pharmaceutically active molecule, characterized in that: The mammalian extracellular vesicles loaded with pharmaceutically active molecules are exosomes, microvesicles or apoptotic bodies loaded with pharmaceutically active molecules prepared by the preparation method described in any one of claims 6 to 8.

11. Use of one or a combination of the mammalian extracellular vesicles according to claim 9 or the mammalian extracellular vesicles loaded with pharmaceutically active molecules according to claim 10 in preparing a pharmaceutical composition.

12. A recombinant mammalian cell, characterized in that The recombinant mammalian cell is the same as the recombinant mammalian cell according to any one of claims 1 to 5, or the recombinant mammalian cell according to any one of claims 6 to 8.

13. A recombinant expression vector system for preparing mammalian extracellular vesicles, characterized in that: The recombinant expression vector system comprises a first recombinant expression vector for expressing the FREE1 protein and a second recombinant expression vector for expressing a fusion protein formed by an extracellular vesicle scaffold protein and a pharmaceutically active molecule or a linker.

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