Exosome for displaying BMP7 on surface as well as preparation method and application of exosome

Abstract

The invention relates to the field of cell biology, and discloses an exosome for displaying BMP7 on the surface as well as a preparation method and application of the exosome. The preparation method comprises the following steps: constructing a fusion gene containing a glycosylation sequence, an HA tag, a BMP7 gene, a Lamp2b gene and a connecting sequence, and the nucleotide sequence of the fusion gene is as shown in SEQ ID NO.1; inserting the fusion gene into an expression cassette of an expression vector to obtain a recombinant expression vector; transferring the recombinant expression vector into a cell which generates the exosome, and screening to obtain a stable BMP7-Lamp2b overexpression cell; and culturing the BMP7-Lamp2b overexpressed cells, collecting the supernatant of the cell culture medium, and purifying to obtain the exosome with the surface displaying BMP7. The prepared exosome with the surface displaying BMP7 can improve the medicine utilization rate and the action time, reduce the medicine use concentration and use frequency, finally reduce the production cost and reduce the infection risk caused by articular cavity injection.

Description

Technical Field

[0001] The present invention relates to the field of cell biology, and in particular to an exosome displaying BMP7 on its surface, and a preparation method and application thereof. Background Art

[0002] Osteoarthritis (OA) is a common asymmetric disease without systemic signs. It is a common and frequently occurring disease in orthopedic clinics. Growth factors can achieve cartilage repair by promoting the proliferation and anabolism of chondrocytes. Transforming growth factors (TGFs) have a wide range of regulatory effects in cartilage and bone tissues, and are therefore widely used in bone and cartilage repair and treatment research. TGFs include transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs), among which TGF-β1, TGF-β3, BMP2, BMP4, BMP7, etc. have shown the effect of alleviating the progression of OA. Among these growth factors, BMP7 has the following characteristics, making it superior to other cartilage regulation-related growth factors, including: 1) BMP7 can inhibit catabolism while promoting anabolism; 2) BMP7 only promotes the synthesis of cartilage extracellular matrix and does not cause hypertrophy; 3) BMP7 can also regenerate normal and functional proteoglycans, and can activate similar anabolic pathways in normal, OA, and aging cartilage tissues; 4) BMP7 can promote the expression of TGF-β, BMPs, and insulin growth factor (IGF-1); 5) BMP7 can inhibit the activity of degradative enzymes by promoting the expression of matrix metalloproteinase tissue inhibitors (TIMPs); 6) BMP7 can also inhibit the expression of inflammatory factors and inhibit inflammatory factor-related pathways. Intra-articular injection of recombinant BMP7 protein can effectively alleviate disease progression in OA animal models, and related phase II clinical trials are also underway.

[0003] Direct administration of human recombinant BMP7 can effectively alleviate the progression of OA, but the problems with this approach are: 1) The half-life of growth factors is short. Studies have shown that the half-life of human recombinant BMP7 in the blood is only 30 minutes, so how to improve the in vivo stability of BMP7 is important for its function; 2) After intra-articular injection, BMP7 can be lost from the joint cavity through lymphatic infiltration, so its residence time in the joint cavity is short, with a half-life of only 1 to 4 hours. Due to the above problems, the direct administration of human recombinant BMP7 to treat OA may have the following problems: 1) low drug utilization; 2) short drug action time, requiring multiple administrations; 3) risk of side effects caused by drug tissue penetration.

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an exosome displaying BMP7 on the surface and a preparation method and application thereof, aiming to solve the problems of low drug utilization rate, short drug action time and risk of side effects in the prior art of using BMP7 to treat osteoarthritis.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, a method for preparing exosomes displaying BMP7 on the surface is provided, comprising the steps of:

[0008] S1. Constructing a fusion gene comprising a glycosylation sequence, an HA tag, a BMP7 gene, a Lamp2b gene and a connecting sequence, wherein the nucleotide sequence of the fusion gene is shown in SEQ ID NO.1;

[0009] S2, inserting the fusion gene into the expression frame of the expression vector to obtain a recombinant expression vector;

[0010] S3, transferring the recombinant expression vector into cells producing exosomes, and screening to obtain stable BMP7-Lamp2b overexpressing cells;

[0011] S4. Cultivating the BMP7-Lamp2b overexpressing cells, collecting the cell culture medium supernatant for purification, and obtaining exosomes displaying BMP7 on the surface.

[0012] In a preferred technical solution, the expression vector is a lentiviral vector.

[0013] In a further preferred technical solution, the lentiviral vector is pLVML-3xHA-MCS-IRES-Puro.

[0014] A preferred technical solution, in the preparation method, step S2 specifically comprises:

[0015] The 5' end of the fusion gene was connected to the 3' end 109 bases downstream of the CMV promoter of pLVML-3xHA-MCS-IRES-Puro, and the 3' end of the fusion gene was connected to the 5' end of the IRES sequence of pLVML-3xHA-MCS-IRES-Puro to obtain a recombinant expression vector through homologous recombination.

[0016] In a preferred technical solution, the cells producing exosomes are HEK293T cells.

[0017] A preferred technical solution, in the preparation method, step S3 specifically comprises:

[0018] After the recombinant expression vector was transferred into the exosome-producing cells for 48 hours, puromycin was used for selection for 6 to 10 days to obtain stable BMP7-Lamp2b overexpressing cells.

[0019] In a second aspect, an exosome displaying BMP7 on its surface is provided, wherein the exosome displaying BMP7 on its surface is prepared by the preparation method described in the first aspect.

[0020] In a third aspect, a method for preparing an osteoarthritis drug is provided.

[0021] Beneficial effects: Compared with human recombinant BMP7 alone, the exosomes displaying BMP7 on the surface prepared by the present invention have higher stability and longer residence time in the joint cavity, thereby improving drug utilization and duration of action, reducing drug concentration and frequency of use, ultimately reducing production costs, and reducing the risk of infection caused by joint cavity injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a fusion pattern diagram of the Lamp2b gene and the BMP7 gene in the fusion gene.

[0023] Figure 2 This is a WB characterization result of HA-BMP7-Lamp2b fusion protein in BMP7-Lamp2b overexpressing cells.

[0024] Figure 3 This is a transmission electron micrograph of surface-displayed BMP7 exosomes.

[0025] Figure 4 This is a tracer analysis of nanoparticles displaying BMP7 exosomes on the surface.

[0026] Figure 5 This is a WB characterization of surface-displayed BMP7 exosomes.

[0027] Figure 6 This is a graph showing the transcriptional regulation of chondrocyte-related genes by BMP7 exosomes on the surface. DETAILED DESCRIPTION

[0028] The present invention provides an exosome displaying BMP7 on its surface, and a preparation method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0029] Exosomes (EVs) are tiny vesicles secreted by cells, with a size of 30 to 150 nm. Exosomes play an important role in intercellular communication by delivering nucleic acids and proteins from mother cells to recipient cells. The good biocompatibility, low immunogenicity and low toxicity of exosomes make them an excellent drug delivery carrier. Through methods such as genetic engineering, small molecules, peptides, nucleic acids, proteins, etc. can be loaded into the cavity or membrane surface of exosomes. After the active molecules loaded in the cavity of exosomes are taken up by the receptor cells, they can play a regulatory function in the cells; while the active molecules anchored on the membrane surface mainly interact with cell membrane proteins or extracellular factors to achieve cell recognition, signal transduction, decoy and other functions. In cartilage tissue, BMP7 interacts with receptor proteins on the surface of chondrocyte membranes to achieve the regulation of downstream signaling pathways. Therefore, by overexpressing BMP7 on the surface of exosomes, when the BMP7 displayed on the surface of exosomes reaches chondrocytes, it can interact with the relevant receptors on the surface of chondrocytes and activate the downstream signaling pathway, thereby achieving OA treatment.

[0030] The present invention realizes the exosome surface display of BMP7 by Lamp2b, thereby improving the stability and joint residence time of BMP7. Lamp2b is the earliest and most commonly used exosome surface modified membrane protein. Since it is highly expressed in exosomes and its N-terminus is located on the membrane surface of the exosomes, by recombinantly expressing the target polypeptide or protein at the N-terminus of Lamp2b, the corresponding polypeptide or protein is overexpressed on the surface of the exosomes, thereby realizing functions such as cell recognition, signal transduction, and bait. Studies have shown that the stability of proteins anchored on the surface of the exosome membrane is limited compared with free proteins. Further introduction of glycosylation sequences in recombinant proteins can significantly improve the stability of surface displayed polypeptides and proteins. At the same time, the overall volume of proteins displayed on the surface of exosomes is larger than that of individual proteins, its lymphatic penetration slows down, and the residence time in the joint cavity becomes longer; moreover, exosomes can also be modified with targeted polypeptides, cationic polymers, etc., to further improve their residence time in the joint cavity.

[0031] Based on the above inventive concept, an embodiment of the present invention provides a method for preparing exosomes displaying BMP7 on the surface, comprising the steps of:

[0032] S1. Constructing a fusion gene comprising a glycosylation sequence, an HA tag, a BMP7 gene, a Lamp2b gene and a connecting sequence, wherein the nucleotide sequence of the fusion gene is shown in SEQ ID NO.1;

[0033] S2, inserting the fusion gene into the expression frame of the expression vector to obtain a recombinant expression vector;

[0034] S3, transferring the recombinant expression vector into cells producing exosomes, and screening to obtain stable BMP7-Lamp2b overexpressing cells;

[0035] S4. Cultivating the BMP7-Lamp2b overexpressing cells, collecting the cell culture medium supernatant for purification, and obtaining exosomes displaying BMP7 on the surface.

[0036] Specifically, the present invention constructs a fusion gene comprising a glycosylation sequence, an HA tag, a BMP7 gene, a Lamp2b gene and a connecting sequence, and its nucleotide sequence is shown in SEQ ID NO.1. Among them, the nucleotide sequence of the glycosylation sequence is shown in SEQ ID NO.2, the nucleotide sequence of the HA tag is shown in SEQ ID NO.3, the nucleotide sequence of the BMP7 gene is shown in SEQ ID NO.4, the nucleotide sequence of the Lamp2b gene is shown in SEQ ID NO.5, the nucleotide sequence of the connecting sequence between the HA tag and the BMP7 gene is GGCGGA, and the nucleotide sequence of the connecting sequence between the BMP7 gene and the Lamp2b gene is shown in SEQ ID NO.6. Further, the present invention inserts the fusion gene into an expression vector, and then transfers it into a cell that produces exosomes, so that the fusion gene expresses HA-BMP7-Lamp2b fusion protein in the cell, thereby obtaining exosomes containing HA-BMP7-Lamp2b fusion protein. Due to the presence of Lamp2b, the HA-BMP7-Lamp2b fusion protein is expressed on the membrane surface of the exosomes. Therefore, the preparation method of the present invention produces an exosome that displays BMP7 on the surface. At the same time, the glycosylation sequence can significantly improve the stability of the HA-BMP7-Lamp2b fusion protein, and the HA tag can be used for the detection, separation and purification of the HA-BMP7-Lamp2b fusion protein.

[0037] In one embodiment, the expression vector is a lentiviral vector.

[0038] In one embodiment, the lentiviral vector is pLVML-3xHA-MCS-IRES-Puro.

[0039] In one embodiment, in the preparation method, step S2 specifically comprises:

[0040] The 5' end of the fusion gene was connected to the 3' end 109 bases downstream of the CMV promoter of pLVML-3xHA-MCS-IRES-Puro, and the 3' end of the fusion gene was connected to the 5' end of the IRES sequence of pLVML-3xHA-MCS-IRES-Puro to obtain a recombinant expression vector through homologous recombination.

[0041] In one embodiment, the exosome-producing cells are HEK293T cells.

[0042] In one embodiment, in the preparation method, step S3 specifically comprises:

[0043] After the recombinant expression vector was transferred into the exosome-producing cells for 48 hours, puromycin was used for selection for 6 to 10 days to obtain stable BMP7-Lamp2b overexpressing cells.

[0044] Based on the same inventive concept, an embodiment of the present invention provides an exosome displaying BMP7 on its surface, wherein the exosome displaying BMP7 on its surface is prepared by the preparation method as described above.

[0045] Based on the same inventive concept, an embodiment of the present invention provides a use of the exosomes displaying BMP7 on the surface as described above in the preparation of osteoarthritis drugs.

[0046] The present invention will be further described below by means of specific examples.

[0047] Experimental materials: Unless otherwise specified, the reagents and materials used in the examples of the present invention are commercially available. Among them, polyethylenimine (PEI) transfection reagent was purchased from Tocris, with the catalog number 7854 / 100, and its specific configuration method is referred to the product description; reduced serum medium Opti-MEM was purchased from ThermoFisher, with the catalog number 51985091; lentiviral vector pLVML-3xHA-MCS-IRES-Puro was purchased from NovoPro Bioscience Inc, with the catalog number V013867.

[0048] Example 1

[0049] This example provides a method for preparing exosomes displaying BMP7 on the surface, and characterizes the prepared exosomes displaying BMP7 on the surface, as follows:

[0050] (1) Gene amplification and fusion: extract mRNA from HEK293T cells using the phenol-chloroform method, obtain cDNA from mRNA using a reverse transcription kit, amplify the BMP7 gene sequence from the cDNA using the PCR method, and recover the BMP7 gene sequence using a DNA gel recovery kit; amplify the Lamp2b gene sequence from the pEGFP-CAP-Lamp2b plasmid using the PCR method, and recover the Lamp2b gene sequence using a DNA gel recovery kit; connect the 5' end of the Lamp2b gene to the 3' end of the BMP7 gene through a linker sequence, add a stop codon to the 3' end of the Lamp2b gene, and add a glycosylation sequence and an HA tag to the 5' end of the BMP7 gene to obtain a fusion gene;

[0051] The nucleotide sequence of the fusion gene is shown in SEQ ID NO.1, wherein the fusion pattern of Lamp2b gene and BMP7 gene is as follows Figure 1 The nucleotide sequence of the glycosylation sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.7; the nucleotide sequence of the HA tag is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.8; the nucleotide sequence of the BMP7 gene is shown in SEQ ID NO.4; the nucleotide sequence of the Lamp2b gene is shown in SEQ ID NO.5; the nucleotide sequence of the connection sequence between the HA tag and the BMP7 gene is GGCGGA; the nucleotide sequence of the connection sequence between the BMP7 gene and the Lamp2b gene is shown in SEQ ID NO.6;

[0052] (2) constructing a recombinant expression vector: inserting the fusion gene into the lentiviral vector pLVML-3xHA-MCS-IRES-Puro, specifically, connecting the 5' end of the fusion gene to the 3' end 109 bases downstream of the CMV promoter of the lentiviral vector pLVML-3xHA-MCS-IRES-Puro, connecting the 3' end of the fusion gene to the 5' end of the IRES sequence of the lentiviral vector pLVML-3xHA-MCS-IRES-Puro, and constructing the recombinant expression vector by homologous recombination, and finally constructing a recombinant expression vector for stably and highly expressing the HA-BMP7-Lamp2b fusion protein encoded by the fusion gene;

[0053] (3) Cell transfection: HEK293T cell lines were cultured in a 3.5 cm cell culture dish until the cell density reached 30%; 4 μg of the correctly sequenced recombinant expression vector was added to a 1.5 ml centrifuge tube containing 100 μl of opti-DMEM and mixed evenly, and then 4 μl of polyethylenimine (PEI, 2 mg / ml) transfection reagent was added thereto and mixed evenly, and allowed to stand at room temperature for 15 min; the above mixture was mixed with freshly prepared DMEM culture medium containing 10% FBS and added to a 3.5 cm culture dish, and the culture dish was placed in an incubator for 12 hours and then the fresh culture medium was replaced;

[0054] (4) Cell screening: 48 hours after cell transfection, overexpressing cells were screened with puromycin at a final concentration of 1.2 μg / ml for 6 to 10 days to obtain stable BMP7-Lamp2b overexpressing cells; untransfected HEK293T cells were used as control cells and GAPDH was used as an internal reference. The expression of HA-BMP7-Lamp2b fusion protein was detected by WB to confirm the establishment of BMP7-Lamp2b overexpressing cells. Figure 2 As shown;

[0055] (5) Collection and purification of exosomes displaying BMP7 on the surface: After culturing BMP7-Lamp2b overexpressing cells to a confluence of 75-80%, the cells were washed three times with phosphate buffered saline (PBS), and the cells were cultured in serum-free DMEM for 24 hours, and the exosome-rich cell culture medium supernatant in the culture dish was collected; the collected cell culture medium supernatant was centrifuged at 10,000 × g for 10 min at room temperature to remove cell debris, and the supernatant was collected; the supernatant was centrifuged at 100,000 × g for 2 hours at 4°C by ultracentrifugation, and the precipitate obtained was the purified exosomes. The purified exosomes were stored in PBS, aliquoted and frozen in a -80°C refrigerator.

[0056] (6) Characterization of exosomes displaying BMP7 on the surface: The extracted exosomes were characterized by transmission electron microscopy, nanoparticle tracing analysis, and Western Blot (WB). From the transmission electron microscopy images, it can be seen that the exosomes displaying BMP7 on the surface present a typical "cup-shaped" vesicle structure, and its particle size distribution is 100-150nm. Figure 3 The results of nanoparticle tracing analysis showed that the particle size of exosomes displaying BMP7 on the surface ranged from 50 to 300 nm, with a peak value of 135 nm. Figure 4 WB results showed that the exosomes displaying BMP7 on the surface overexpressed HA-BMP7-Lamp2b fusion protein with HA tag and expressed exosome marker protein CD63, see Figure 5 .

[0057] Example 2

[0058] This example detects the effect of the exosomes displaying BMP7 prepared in Example 1 on the expression level of chondrocyte-related genes, as follows:

[0059] (1) Cell treatment: Human chondrosarcoma cells SW1353 were passaged in six-well plates to a total of approximately 100,000 cells per well. After overnight culture, the cells were treated with interleukin-1β (IL-1β) at a final concentration of 10 ng / ml for 24 h. The cells were then treated with surface-expressing BMP7 exosomes at final concentrations of 5 and 10 μg / ml, respectively, for another 24 h.

[0060] (2) Real-time fluorescence quantitative polynucleotide chain reaction (QPCR) to identify the expression level of related genes: The culture medium was discarded, the cells were washed twice with phosphate buffered saline (PBS), the RNA in the cells was extracted using the phenol-chloroform method, cDNA was obtained by reverse transcription, and the expression levels of genes such as Col2a1, FGF2, IL6, MMP13, SOX9, and Survivin in chondrocytes were analyzed using QPCR with GAPDH as the internal reference. From the QPCR results, it can be seen that the surface-displayed BMP7 exosomes can promote the expression of anabolism-related genes SOX9 and Col2a1, the expression of proliferation-related gene Survivin, and the expression of migration-related gene FGF2 in chondrocytes, and inhibit the expression of catabolism-related gene MMP13 and inflammation-related gene IL6 in chondrocytes, see Figure 6 .

[0061] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing exosomes displaying BMP7 on the surface, characterized in that: Includes steps: S1. Constructing a fusion gene comprising a glycosylation sequence, an HA tag, a BMP7 gene, a Lamp2b gene and a connecting sequence, wherein the nucleotide sequence of the fusion gene is shown in SEQ ID NO.1; S2, inserting the fusion gene into the expression frame of the expression vector to obtain a recombinant expression vector; S3, transferring the recombinant expression vector into cells producing exosomes, and screening to obtain stable BMP7-Lamp2b overexpressing cells; S4. Cultivating the BMP7-Lamp2b overexpressing cells, collecting the cell culture medium supernatant for purification, and obtaining exosomes displaying BMP7 on the surface.

2. The preparation method according to claim 1, characterized in that: The expression vector is a lentiviral vector.

3. The preparation method according to claim 2, characterized in that: The lentiviral vector is pLVML-3xHA-MCS-IRES-Puro.

4. The preparation method according to claim 3, characterized in that: In the preparation method, step S2 specifically comprises: The 5' end of the fusion gene was connected to the 3' end 109 bases downstream of the CMV promoter of pLVML-3xHA-MCS-IRES-Puro, and the 3' end of the fusion gene was connected to the 5' end of the IRES sequence of pLVML-3xHA-MCS-IRES-Puro to obtain a recombinant expression vector through homologous recombination.

5. The preparation method according to claim 1, characterized in that: The cells producing exosomes are HEK293T cells.

6. The preparation method according to claim 1, characterized in that: In the preparation method, step S3 specifically comprises: After the recombinant expression vector was transferred into the exosome-producing cells for 48 hours, puromycin was used for selection for 6 to 10 days to obtain stable BMP7-Lamp2b overexpressing cells.

7. An exosome displaying BMP7 on its surface, characterized in that: The exosomes displaying BMP7 on the surface are prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the exosomes displaying BMP7 on the surface as claimed in claim 7 in the preparation of osteoarthritis drugs.

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