A method for selectively capturing functional exosomes and its purification and application

Abstract

The application provides a method for selectively capturing functional exosomes and a purification method and application thereof, and relates to the technical field of exosome purification. The application constructs a fusion protein with cutting characteristics and a corresponding expression vector, and modifies an exosome donor cell by using the vector. The exosome donor cell simultaneously overexpresses a therapeutic mRNA carrying MS2. The application constructs a cuttable fusion protein expression vector, so that the loading of a nucleic acid drug in an exosome and the presentation of a specific epitope on the surface of the exosome are realized in the same exosome, thereby facilitating the loading of an exosome mRNA drug and subsequent selective enrichment. The purification method is relatively low in cost and convenient.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome purification, and specifically relates to a method for selectively capturing functional exosomes and a purification method and application thereof. Background Art

[0002] Exosomes are nanoscale extracellular vesicles measuring 30-150 nm released by cells. As delivery vehicles, exosomes are an advanced platform for drug and gene delivery due to their unique properties, including inherent stability, low immunogenicity, and good cell / tissue penetration. To date, most studies have isolated all exosomes as therapeutic delivery vehicles and evaluated their therapeutic efficacy without considering exosome heterogeneity. However, not all exosomes contain the same abundance of a given substance. Based on this theoretical foundation, methods for isolating exosomes are crucial. Current methods, including ultracentrifugation and exosome isolation kits, simply isolate large numbers of exosomes without distinguishing specific subpopulations. Furthermore, immunocapture methods isolate specific exosome subpopulations using specific protein markers (such as CD63). Exosomes isolated by this selective approach also exhibit heterogeneity in content, ignoring the fact that different subpopulations may share common markers. None of these methods achieves precise separation of heterogeneous exosomes at a homogeneous level. Therefore, the development of a universal, cost-effective, and highly sensitive method for purifying functionally specific exosome subpopulations is a priority. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a method and application for selectively capturing functional exosomes and purifying the same, which can efficiently enrich the target (therapeutic) exosomes from the heterogeneous population of exosomes.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a group of fusion proteins with cleavage properties, wherein the fusion proteins include a first fusion protein and a second fusion protein;

[0006] The structure of the first fusion protein includes, from N-terminus to C-terminus: a Flag tag, a thrombin-specific cleavage site, an exosome transmembrane protein, cathepsin L, and MCP;

[0007] The structure of the second fusion protein includes LDLR and MS2 from N-terminus to C-terminus.

[0008] Preferably, the nucleotide sequence of the gene encoding the Flag tag is shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding the thrombin-specific cleavage site is shown in SEQ ID NO.2, the nucleotide sequence of the gene encoding the exosomal transmembrane protein is shown in SEQ ID NO.3, the nucleotide sequence of the gene encoding cathepsin L is shown in SEQ ID NO.4, the nucleotide sequence of the gene encoding MCP is shown in SEQ ID NO.5, the nucleotide sequence of the gene encoding LDLR is shown in SEQ ID NO.6, and the nucleotide sequence of the gene encoding MS2 is shown in SEQ ID NO.7.

[0009] The present invention provides a group of expression vectors comprising the gene encoding the above-mentioned fusion protein.

[0010] Preferably, the basic vector of the expression vector includes pcDNA3.1(-).

[0011] The present invention also provides the use of the above-mentioned fusion protein or the above-mentioned expression vector in modifying exosomes.

[0012] The present invention also provides a method for modifying exosomes, comprising the following steps: mixing the above-mentioned expression vector with a transfection kit and a serum DMEM culture medium for co-incubation;

[0013] After the co-incubation solution and cells were mixed and allowed to stand for 4 to 6 hours, the culture medium was replaced with serum-free and double-antibody-free DMEM for culture. After 48 hours of culture, the cell supernatant was collected and the exosomes were extracted to obtain the modified exosomes.

[0014] Preferably, the volume ratio of the total mass of the expression vector to the transfection reagent is 6-10 μg:16-20 μL.

[0015] The present invention also provides a method for purifying exosomes, comprising the following steps: incubating the modified exosomes obtained by the above modification method with anti-Flag magnetic beads to obtain a magnetically labeled sample;

[0016] Under the action of the magnetic field, the magnetically labeled sample is passed through the collection column. After the magnetic field is turned off, it is eluted with PBS and the eluate is collected;

[0017] After the eluate is incubated with thrombin, the incubation solution is passed through under the action of a magnetic field to collect the eluate containing purified exosomes without magnetic beads.

[0018] Preferably, after collecting the eluate of purified exosomes without magnetic beads, the process further comprises discarding the supernatant after centrifugation and acidifying.

[0019] The present invention also provides the application of the above purification method in exosome mRNA drug loading and selective enrichment.

[0020] Beneficial Effects: The present invention provides a set of fusion proteins with cleavage properties and corresponding expression vectors. By constructing a cleavable fusion protein expression vector, exosome loading of nucleic acid drugs and surface presentation of specific epitopes can be achieved within the same exosome, thereby facilitating exosomal mRNA drug loading and subsequent selective enrichment. To further improve exosomal mRNA loading efficiency, the present invention incorporates an MS2-MCP system into exosomes during the construction of the fusion protein, selectively enriching the target mRNA and preventing mRNA degradation.

[0021] The fusion protein expression vector constructed using the present invention can efficiently purify and enrich therapeutic exosomes. On the outside of the exosomes, the Flag tag protein and the thrombin cleavable site are sequentially fused to the N-terminus of the exosome transmembrane protein (PTGFRN). Therefore, the anti-Flag magnetic beads can accurately identify the Flag tag protein outside the exosome membrane and specifically capture the target exosomes. After the specifically captured target exosomes are collected by the magnetic field, the separation MCP fusion of the magnetic beads and the target exosomes is achieved by co-incubation of thrombin with the target exosomes. MCP can specifically bind to the target mRNA containing MS2, thereby improving the loading efficiency of the target mRNA. Giving the inside of the exosomes an acidic environment allows cathepsin L (CTSL) to self-hydrolyze and separate from PTGFRN, allowing the target mRNA to freely and efficiently function in the recipient cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result diagram of the expression of extracted exosomes;

[0023] Figure 2 This is the western blot test result of the extracted, purified and acidified exosomes. DETAILED DESCRIPTION

[0024] The present invention provides a group of fusion proteins with cleavage properties, wherein the fusion proteins include a first fusion protein and a second fusion protein;

[0025] The structure of the first fusion protein includes, from N-terminus to C-terminus: a Flag tag, a thrombin-specific cleavage site, an exosome transmembrane protein, cathepsin L, and MCP;

[0026] The structure of the second fusion protein includes LDLR and MS2 from N-terminus to C-terminus.

[0027]

[0028] The present invention provides a group of expression vectors comprising the gene encoding the above-mentioned fusion protein.

[0029] The base vector of the expression vector of the present invention preferably comprises pcDNA3.1(-), and the genes encoding the fusion proteins are respectively inserted into the multiple cloning site of the pcDNA3.1(-). In an embodiment of the present invention, the fusion proteins formed by sequential ligation are preferably inserted between the Nhel and EcoRV restriction sites in the 5' to 3' direction.

[0030] The present invention constructs a cleavable fusion protein expression vector, so that exosome loading of nucleic acid drugs and surface presentation of specific epitopes are achieved in the same exosome, thereby facilitating exosome mRNA drug loading and subsequent selective enrichment.

[0031] The present invention also provides the use of the above-mentioned fusion protein or the above-mentioned expression vector in modifying exosomes.

[0032] The present invention also provides a method for modifying exosomes, comprising the following steps: mixing the above-mentioned expression vector with a transfection kit and a serum DMEM culture medium for co-incubation;

[0033] After the co-incubation solution and cells were mixed and allowed to stand for 4 to 6 hours, the culture medium was replaced with serum-free and double-antibody-free DMEM for culture. After 48 hours of culture, the cell supernatant was collected and the exosomes were extracted to obtain the modified exosomes.

[0034] The total mass of the expression vector of the present invention and the volume ratio of the transfection reagent are preferably 6-10 μg:16-20 μL, and the mass ratio of the expression vector containing the first fusion protein to the expression vector containing the second fusion protein is preferably 1:1. In the present invention, the plasmids of the two expression vectors are preferably incubated with the transfection reagent (Invitrogen, USA) in serum-free DMEM medium (Gibco, USA) for 15 minutes before adding to the culture dish.

[0035] The present invention preferably replaces the culture medium in the dish with serum-free and anti-antibody-free DMEM medium after standing in the culture dish for 4 to 6 hours, and collects the cell supernatant after 48 hours. The present invention does not specifically limit the method for extracting exosomes from the cell supernatant, but preferably includes low-speed centrifugation (500g, 10 min) at 4°C to remove dead cells and cell debris, followed by high-speed centrifugation (2000g, 10 min) to remove biopolymers and apoptotic bodies, and then centrifugation at 10,000g for 30 minutes to remove large vesicles, and finally ultracentrifugation (100,000g, 2h) to obtain exosomes.

[0036] The present invention also provides a method for purifying exosomes, comprising the following steps: incubating the modified exosomes obtained by the above modification method with anti-Flag magnetic beads to obtain a magnetically labeled sample;

[0037] Under the action of the magnetic field, the magnetically labeled sample is passed through the collection column. After the magnetic field is turned off, it is eluted with PBS and the eluate is collected;

[0038] After the eluate is incubated with thrombin, the incubation solution is passed through under the action of a magnetic field to collect the eluate containing purified exosomes without magnetic beads.

[0039] The present invention preferably collects the specifically captured target exosomes through a magnetic field, and then separates the magnetic beads from the target exosomes by co-incubating thrombin with the target exosomes; within the exosome cavity, the C-terminus of the exosome transmembrane protein PTGFRN is fused with cathepsin L and MCP in sequence; MCP can specifically bind to the target mRNA containing MS2, thereby improving the loading efficiency of the target mRNA.

[0040] In the present invention, the exosomes obtained above are preferably incubated with anti-Flag magnetic beads (MedChemExpress, USA) at room temperature for 1 hour. Simultaneously, a collection column is placed in a magnetic field and rinsed with 1 mL of PBS. The magnetically labeled sample is then added to the collection column and allowed to pass through. After the collection column is removed from the magnetic field, it is rinsed again with 1 mL of PBS, and the eluted purified exosomes are collected. The purified exosomes are then incubated with thrombin (Solarbio, China) in Tris-HCl buffer (pH 8.0) for 30 minutes. The exosomes are then added back to a new column placed in the magnetic field, and the anti-Flag magnetic beads adhering to the column are discarded. The eluate containing the purified exosomes, which is free of magnetic beads, is then collected.

[0041] After collecting the eluate of purified exosomes without magnetic beads, the present invention preferably further comprises centrifugation, discarding the supernatant, and acidification. The centrifugation preferably comprises centrifugation at 12,000 g for 20 minutes. After centrifugation, the supernatant is discarded, PBS (pH 5) is added to the centrifuge tube, and the exosomes are homogenized by pipetting. The tube is then placed in an electroporation cuvette, pulsed with 350 V, and allowed to stand at 4°C for 30 minutes. The acidification procedure creates an acidic environment within the exosomes, enabling cathepsin L (CTSL) to self-hydrolyze and separate from PTGFRN, allowing the target mRNA to freely and efficiently function in recipient cells.

[0042] The present invention also provides the application of the above purification method for loading and selective enrichment of exosome mRNA drugs. The application of the present invention is preferably the same as the above method and will not be repeated here.

[0043] The following examples provide a detailed description of a method for selectively capturing functional exosomes and its purification method and application provided by the present invention, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1 Construction of expression vector

[0045] Entrust Nanjing GenScript Biotech Co., Ltd. to complete the construction of expression vector

[0046] 1: Expression vector 1: Select Nhel and EcoRV as restriction sites, and pcDNA3.1(-) as the vector.

[0047] From the 5' end to the 3' end, the Flag tag protein, Thrombin cleavable site, PTGFRN, CTSL and MCP are connected in sequence;

[0048] 2: Expression vector 2: Select Nhel and EcoRV as restriction sites, and pcDNA3.1(-) as the vector.

[0049] LDLR and MS2 are connected sequentially from the 5' end to the 3' end.

[0050] 3. 5 μg of expression vector 1 and 5 μg of expression vector 2 were incubated with 20 μL of transfection reagent (Invitrogen, USA) in serum-free DMEM medium (Gibco, USA) for 15 minutes before addition to the culture dish. After 4-6 hours, the culture medium in the dish was completely replaced with serum-free DMEM medium without dual antibody, and the cell supernatant was collected after 48 hours.

[0051] Dead cells and cell debris were removed by low-speed centrifugation (500 g, 10 min) at 4°C, followed by high-speed centrifugation (2000 g, 10 min) to remove biopolymers and apoptotic bodies. Large vesicles were removed by centrifugation at 10,000 g for 30 min, and exosomes were obtained by ultracentrifugation (100,000 g, 2 h).

[0052] Example 2 Extraction of exosomes for qRT-PCR detection

[0053] From the exosomes obtained in Example 1, total RNA was extracted using Tripure Isolation Reagent (Invitrogen, USA) according to the instructions provided by the reagent manufacturer.

[0054] The mRNA was reverse transcribed using the PrimeScript First-Strand cDNA Synthesis Kit (Takara, China).

[0055] Gene expression was analyzed using PrimeScript RT MasterMix (Roche, Switzerland).

[0056] FastStart Essential DNA Green Master was used to establish the qPCR reaction system. The data were collected and analyzed using the Roche LightCycler 96 qPCR system (Roche). GAPDH was used as an internal control. -ΔΔCt Method to calculate relative expression, primer pair information:

[0057] MouseLdlr:

[0058] Forward (SEQ ID NO.8): 5'-TGACTCAGACGAACAAGGCTG-3';

[0059] Reverse (SEQ ID NO.9): 5'-CTAACTAAACACCAGACAGAGGC-3';

[0060] Mouse Gapdh:

[0061] Forward (SEQ ID NO.10): 5'-AGGTCGGTGTGAACGGATTTG-3';

[0062] Reverse (SEQ ID NO. 11): 5'-TGTAGACATGTAGTTGAGGTCA-3'.

[0063] Reaction system: 20 μL (9 μL FastStart Essential DNA Green Master, 1 μL primer, 1 μL cDNA, 9 μL water)

[0064] Reaction program: 95°C for 2 min, 95°C for 10 s, 60°C for 30 s, 45 cycles.

[0065] Expression level Figure 1 As shown, exosomes not only successfully loaded the target mRNA, but also selectively enriched the target mRNA.

[0066] Example 3:

[0067] Extract, purify and acidify exosomes and perform western blot detection

[0068] 1. Purification

[0069] The exosomes obtained in Example 1 were incubated with anti-Flag magnetic beads (MedChemExpress, USA) at room temperature for 1 hour. The collection column was placed in a magnetic field and rinsed with 1 mL of PBS. The magnetically labeled sample was then added to the collection column and allowed to pass through. After the collection column was removed from the magnetic field, it was re-rinsed with 1 mL of PBS and the eluted purified exosomes were collected. The purified exosomes were then incubated with thrombin (Solarbio, China) in Tris-HCl buffer (pH = 8.0) for 30 minutes. The exosomes were then added back to a new column placed in the magnetic field. The anti-Flag magnetic beads adhered to the column were discarded, and the eluate containing the purified exosomes, which did not contain the magnetic beads, was collected.

[0070] 2. Acidification of exosomes

[0071] The purified exosomes were centrifuged and the supernatant was discarded. PBS (pH = 5) was added to the centrifuge tube to evenly distribute the exosomes. The tube was then placed in an electric rotating cup, pulsed with a voltage of 350 V, and then left to stand at 4°C for 30 min.

[0072] 3. Western blot detection

[0073] The exosomes were lysed in 60 μL of RIPA buffer (Beyotime, China) after centrifugation. The exosomes were pipetted until completely dissolved and then lysed on ice for 30 min. Protein was quantified by serial dilution (Thermo, USA) and protein samples were prepared.

[0074] After preparing a 12% SDS-PAGE gel, the exosome protein sample was added for electrophoresis. The lysate was separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, USA). The membrane was blocked with 3% skim milk in TBST for 1 hour and incubated with anti-GAPDH polyclonal antibody (D110016-0100, BBI Life Sciences), anti-MCP (ABE76, Merck Millipore), and anti-CTSL (ab200738, Abcam) primary antibodies at 4°C overnight. After washing with TBST, incubate with secondary antibody at room temperature for 1 hour and wash away excess secondary antibody with TBST again. GAPDH was used as an internal control, and the blot was visualized using ECL luminescent solution (Thermo, USA). The visualization results are shown in Figure 2. Figure 2 Shown: Exo Ctrl Exosomes are untransfected HEK293T cell exosomes. Flag / Ldlr pExo is the exosome of HEK293T cells after co-transfection of expression vector 1 and expression vector 2. Flag / LdlrProtein immunoblotting analysis of HEK293T cell exosomes co-transfected with expression vector 1 and expression vector 2 after purification and acidification showed that the target gene Ldlr carried by the purified and acidified exosomes had a higher expression level in the cells.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Air Force Medical University of the Chinese People's Liberation Army <120> A method for selectively capturing functional exosomes and its purification and application <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> 72 <212> DNA <213> Artificial Sequence <400> 1 gactacaaag acgatgacga caaggactac aaagacgatg acgacaagga ctacaaagac 60 gatgacgaca ag 72 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <400> 2 ggaggactag taccccgcgg aagcggagga 30 <210> 3 <211> 579 <212> DNA <213> Artificial Sequence <400> 3 ggtcctatat ttaatgcttc tgtgcattca gacacaccat cagtaattcg gggagatctg 60 atcaaattgt tctgtatcat cactgtcgag ggagcagcac tggatccaga tgacatggcc tttgatgtgt cctggtttgc ggtgcactct tttggcctgg acaaggctcc tgtgctcctg 180 tcttccctgg atcggaaggg catcgtgacc acctcccgga gggactgga gagcgacctc 240 agcctggagc gcgtgagtgt gctggaattc ttgctgcaag tgcatggctc cgaggaccag 300 gactttggca actactactg ttccgtgact ccatgggtga agtcaccaac aggttcctgg cagaaggagg cagagatcca ctccaagccc gtttttata ctgtgaagat ggatgtgctg aacgccttca agtatccctt gctgatcggc gtcggtctgt ccacggtcat cgggctcctg 480 tcctgtctca tcgggtactg cagctcccac tggtgttgta agaaggaggt tcaggagaca 540 cggcgcgagc gccgcaggct catgtcgatg gagatggac 579 <210> 4 <211> 948 <212> DNA <213> Artificial Sequence <400> 4 actctaacat ttgatcacag tttagaggca cagtggacca agtggaaggc gatgcacaac attach gcatgaatga agaaggatgg aggagagcag tgtgggaga gaacatgaag atgattgac tgcacaatca ggaatacagg gaagggaac acagcttcac aatggccatg aacgcctttg gagacatgac cagtgaagaa ttcaggcagg tgatgaatgg ctttcaaaac cgtaagccca ggaagggga agtgttccag gaacctctgt tttatgaggc ccccagatct 360. gtggattgga gagagaaagg ctacgtgact cctgtgaaga atcagggtca gtgtggttct tgttgggctt ttagtgctac tggtgctctt gaggacaga tgttccgga aactgggagg 420 cttatctcac tgagtgagca gaatctggta gactgctctg ggcctcaagg caatgaaggc tgcaatggtg gcctaatgga ttatgctttc cagtatgttc aggataatgg aggcctggac 540 tctgaggaat cctatccata tgaggcaaca gagaatcct gtaagtacaa tcccaagtat tctgttgcta atgacaccgg ctttgtggac atccctaagc aggagaaggc cctgatgaag 660 gcagttgcaa ctgtggggcc catttctgtt gctattgatg caggtcatga gtccttcctg 720 ttctaaag aaggcattta ttttgagcca gactgtagca gtgaagacat ggatcatggt gtgctggtgg ttggctacgg atttgaaagc acagaatcag father atttggctg gtgaagaaca gctggggtga agaatggggc atgggtggct acgtaaagat ggccaaagac 900 cggagaaacc attgtggaat tgcctcagca gccagctacc ccactgtg 948 <210> 5 <211> 387 <212> DNA <213> Artificial Sequence <400> 5 gcttcaaact ttactcagtt cgtgctcgtg gacaatggtg ggacagggga tgtgacagtg 60 gctccttcta atttcgctaa tggggtggca gagtggatca gctccaactc acggagccag 120 gcctacaagg tgacatgcag cgtcaggcag tctagtgccc agaagagaaa gtataccatc 180 aaggtggagg tccccaaagt ggctacccag acagtgggcg gagtcgaact gcctgtcgcc 240 gcttggaggt cctacctgaa catggagctc actatcccaa ttttcgctac caattctgac 300 tgtgaactca tcgtgaaggc aatgcagggg ctcctcaaag acggtaatcc tatcccttcc 360 [[ID=२५]] gccatcgccg ctaactcagg tatctac 387 <210> 6 <211> 2433 <212> DNA <213> Artificial Sequence <400> 6 atgagcaccg cggatctgat gcgtcgctgg gtcatcgccc tgctcctggc tgctgccgga 60 gttgcagcag aagactcatg cagcaggaac gagttccagt gtagagacgg aaaatgcatc 120 gctagcaagt gggtgtgcga tggcagcccc gagtgcccgg atggctccga tgagtcccca 180 gagacatgca tgtctgtcac ctgtcagtcc aatcaattca gctgtggagg ccgtgtcagc 240 cgatgcattc ctgactcctg gagatgtgat ggacaggtag actgtgaaaa tgactcagac 300 gaacaaggct gtccccccaa gacgtgctcc caggatgact tccgatgcca ggatggcaag 360 tgcatctccc cgcagtttgt gtgtgatgga gaccgagatt gcctagatgg ctctgatgag 420 gcccactgcc aggccaccac ttgtggcccc gcccacttcc gctgcaactc atccatatgc 480 atccccagtc tttgggcctg cgacggggat gtcgactgtg ttgacggctc cgatgagtgg 540 ccacagaact gccagggccg agacacggcc tccaaaggcg ttagcagccc ctgctcctcc 600 ctggagttcc actgtggtag cagtgagtgt atccatcgca gctgggtctg tgacggcgag 660 gcagactgca aggacaagtc agatgaggag cactgcgcgg tggccacctg ccgacctgat 720 gaattccagt gtgcagatgg ctcctgcatt cacggtagcc gccagtgtga ccgtgaacat 780 gactgcaagg acatgagcga cgagctcggc tgcgtcaatg tgacacagtg tgatggcccc 840 aacaagttca agtgtcacag tggggagtgc atcagcttgg acaaggtgtg cgactccgcc 900 cgcgactgcc aggactggtc ggatgagccc atcaaggagt gcaagaccaa cgagtgtttg 960 gacaacaatg gtggctgttc ccacatctgc aaggacctca agattggctc tgagtgcctg 1020 tgtcccagcg gcttccggtt ggtggacctc cacaggtgtg aagatattga cgagtgtcag 1080 gagccagaca cctgcagcca gctctgtgtg aacctggaag gcagctacaa gtgtgagtgc 1140 caggccggct tccacatgga cccacacacc agggtctgca aggctgtggg ctccataggc 1200 tatctgctct tcaccaaccg ccacgaggtc cggaagatga ccctggaccg cagcgagtac 1260 accagtctgc tccccaacct gaagaatgtg gtggctctcg acacggaggt gaccaacaat 1320 agaatctact ggtccgacct gtcccaaaaa aagatctaca gcgccctgat ggaccaggcc 1380 cctaacttgt cctacgacac catcatcagt gaggacctgc atgcccctga cgggctggcg 1440 gtagactgga tccaccgcaa catctactgg acagattcag tcccaggcag cgtatctgtg 1500 gctgacacca agggcgtaaa gaggaggaca ctgttccaag aggcagggtc cagacccaga 1560 gccatcgtag tggaccctgt gcatggcttc atgtactgga cagattgggg aacacccgcc 1620 aagatcaaga aagggggttt gaatggtgtg gacatccact cactggtgac cgaaaacatc 1680 cagtggccaa atggcatcac actagatctt tccagtggcc gtctctattg ggttgattcc 1740 aaactccact ctatctccag catcgatgtc aatgggggca atcggaaaac cattttggag 1800 gatgagaacc ggctggccca ccccttctcc ttggccatct atgaggacaa agtgtattgg 1860 acagatgtca taaacgaagc cattttcagt gccaatcgac tcacgggttc agatgtgaat 1920 ttggtggctg aaaacctctt gtccccggag gacattgtcc tgttccacaa ggtcacacag 1980 cctagagaag tcgacactgt actgaccacc caggggacat ccgccgtccg gcctgtggtc 2040 accgcatcag ctaccaggcc accgaagcac agtgaggatc tctcagctcc cagtactcct 2100 aggcagcctg tggacacccc agggctcagc acagtggcgt cagtgacagt gtcccaccaa 2160 gtccagggtg acatggctgg cagagggaat gaggagcagc cacatggtat gaggttcctg 2220 tccatcttct tccctattgc actggttgcc ctccttgtcc ttggggccgt cctgctgtgg 2280 aggaactggc ggctgaagaa catcaacagc ataaactttg acaacccagt ctaccagaag 2340 accacagagg acgagctcca catttgccga agccaggatg gctataccta cccctcaaga 2400 cagatggtca gcctggagga cgatgtggca tga 2433 <210> 7 <211> 75 <212> DNA <213> Artificial Sequence <400> 7 ctagaaaaca tgaggatcac ctcatgtctg caggtcgatc tagaaaaaca tgaggatcac 60 ctcatgtctg caggt 75 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <400> 8 tgactcagac gaacaaggct g 21 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <400> 9 ctaactaaac accagacaga ggc 23 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <400> 10 aggtcggtgt gaacggattt g 21 <210> 11 <211> twenty three <212> DNA <213> Artificial Sequence <400> 11 tgtagaccat gtagttgagg tca 23

Claims

1. A loading and delivery system with cutting properties, characterized in that: Including fusion protein and mRNA loading delivery structures; The structure of the fusion protein from N-terminus to C-terminus is: Flag tag, thrombin specific cleavage site, exosome transmembrane protein, cathepsin L and MCP; From the 5' end to the 3' end of the mRNA loading delivery structure, there are: LDLR and MS2; The nucleotide sequence of the gene encoding the Flag tag is shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding the thrombin-specific cleavage site is shown in SEQ ID NO.2, the nucleotide sequence of the gene encoding the exosomal transmembrane protein is shown in SEQ ID NO.3, the nucleotide sequence of the gene encoding cathepsin L is shown in SEQ ID NO.4, the nucleotide sequence of the gene encoding MCP is shown in SEQ ID NO.5, the nucleotide sequence of the gene encoding LDLR is shown in SEQ ID NO.6, and the nucleotide sequence of the gene encoding MS2 is shown in SEQ ID NO.

7.

2. A group of expression vectors comprising the gene encoding the delivery system according to claim 1.

3. The expression vector according to claim 2, characterized in that The basic vector of the expression vector includes pcDNA3.1(-).

4. Use of the loading and delivery system according to claim 1 or the expression vector according to claim 2 or 3 in modifying exosomes.

5. A method for modifying exosomes, characterized in that: The method comprises the following steps: mixing the expression vector according to claim 2 or 3 with a transfection kit and a serum DMEM culture medium for co-incubation; After the co-incubation solution and cells were mixed and allowed to stand for 4 to 6 hours, the culture medium was replaced with serum-free and double-antibody-free DMEM for culture. After 48 hours of culture, the cell supernatant was collected and the exosomes were extracted to obtain the modified exosomes.

6. The modification method according to claim 5, characterized in that The volume ratio of the total mass of the expression vector to the transfection reagent is 6-10 μg:16-20 μL.

7. A method for purifying exosomes, characterized in that: The following steps are involved: incubating the modified exosomes obtained by the modification method of claim 5 or 6 with anti-Flag magnetic beads to obtain a magnetically labeled sample; Under the action of the magnetic field, the magnetically labeled sample is passed through the collection column. After the magnetic field is turned off, it is eluted with PBS and the eluate is collected; After the eluate is incubated with thrombin, the incubation solution is passed through under the action of a magnetic field to collect the eluate containing purified exosomes without magnetic beads.

8. The purification method according to claim 7, characterized in that After collecting the eluate of purified exosomes without magnetic beads, the supernatant is discarded after centrifugation and acidified.

9. Application of the purification method according to claim 7 or 8 in exosome mRNA drug loading and selective enrichment, characterized in that: The exosomal mRNA drug is LDLR.

Images