Vesicle for transporting target protein as well as preparation method and application of vesicle

Abstract

The invention discloses an exogenous gene delivery method, which comprises the following steps of: transfecting vesicles by using a fusion expression vector or a co-expression vector of an ORF3 protein gene of hepatitis E virus and an exogenous gene to generate cells, purifying cell culture supernatant by using a differential ultracentrifugation method to obtain vesicles, transfecting vesicle recipient cells by using the vesicles, and transferring the vesicles to obtain the exogenous gene. And further transferring an exogenous gene product into recipient cells. The invention also discloses a gene editing method, which comprises the following steps of: preparing a carrier which contains the fusion gene of the ORF3 protein of the hepatitis E virus and the Cas9 protein, inserting independently expressed sgDNA, transfecting vesicles by using the carrier to generate cells, purifying cell culture supernatant by using a differential ultracentrifugation method to obtain the vesicles, transfecting vesicle recipient cells by using the vesicles, and obtaining the gene editing product. And target genes corresponding to the sgDNA are edited in recipient cells. The method disclosed by the invention has an application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a vesicle for transporting target proteins, its preparation method and application. Background Technology

[0002] Current target protein delivery systems are mainly lipid particle delivery systems, which suffer from limited delivery capacity and high cost. To address these technical challenges, there is an urgent need to develop new target protein delivery systems.

[0003] The hepatitis E virus ORF3 protein (HEV-ORF3 protein for short) is a multifunctional small protein encoded by hepatitis E virus (HEV) of the Hepeviridae family. Previous studies have suggested that the HEV-ORF3 protein can package HEV viral particles into vesicles and mediate the release of vesicle-encapsulated viral particles, and that these vesicle-encapsulated viral particles have the ability to infect different types of cells.

[0004] However, there are no reports of HEV-ORF3 protein carrying exogenous protein secretion, no reports of HEV-ORF3 protein bringing its fused protein into vesicles (or exosomes), and no reports of HEV-ORF3 protein bringing its fused CAS9 protein into vesicles (or exosomes) and using gRNA to edit other genes. Such applications could unlock the value of targeted delivery of exogenous proteins and targeted gene editing. Summary of the Invention

[0005] The first aspect of the present invention provides a biomaterial, said biomaterial being any one of the following M1, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, M12 and M13;

[0006] M1: Genetic engineering vector

[0007] The genetic engineering vector is a first genetic engineering vector, a second genetic engineering vector, or a third genetic engineering vector; the first genetic engineering vector contains a first gene expression cassette, which contains the coding sequence of the ORF3 protein of hepatitis E virus and a foreign gene insertion site; after the foreign gene is inserted at the foreign gene insertion site, the fusion protein expressed by the first gene expression cassette includes the ORF3 protein of hepatitis E virus and the protein encoded by the foreign gene; the second genetic engineering vector contains a second gene expression cassette and a third gene expression cassette; the second gene expression cassette can express the ORF3 protein gene of hepatitis E virus; the third gene expression cassette... The expression cassette contains a foreign gene insertion site for inserting and expressing the foreign gene. The third gene engineering vector contains a fourth and a fifth gene expression cassette. The fusion protein expressed by the fourth gene expression cassette includes the ORF3 protein of hepatitis E virus and the Cas9 protein suitable for the CRISPR / Cas9 system. The fifth gene expression cassette contains an sgDNA insertion site corresponding to the target sequence suitable for the CRISPR / Cas9 system for targeting the target gene. After the sgDNA is inserted into the insertion site, the fifth gene expression cassette can transcribe sgRNA for targeting the target gene.

[0008] M2: Recombinant gene engineering vector

[0009] The recombinant gene engineering vector is a first recombinant gene engineering vector, a second recombinant gene engineering vector, or a third recombinant gene engineering vector; the exogenous gene is inserted into the exogenous gene insertion site of the first gene engineering vector to form the first recombinant gene engineering vector; the exogenous gene is inserted into the exogenous gene insertion site of the second gene engineering vector to form the second recombinant gene engineering vector; the sgDNA is inserted into the insertion site of the third gene engineering vector to form the third recombinant gene engineering vector.

[0010] M3: Genetic engineering vector combination

[0011] The genetic engineering vector combination includes a lentiviral vector plasmid, a lentiviral packaging helper plasmid, and a lentiviral envelope plasmid; the lentiviral vector plasmid is selected from the genetic engineering vector described in M1 or the recombinant genetic engineering vector described in M2.

[0012] M4: Fusion protein

[0013] The fusion protein is the fusion protein expressed by the first gene expression cassette in the first recombinant gene engineering vector;

[0014] M5: RNA

[0015] The RNA can be translated to obtain the fusion protein described in M4;

[0016] M6: Protein Combination

[0017] The protein combination is a combination of the protein expressed by the second gene expression cassette in the second recombinant gene engineering vector and the protein expressed by the third gene expression cassette.

[0018] M7: RNA combination

[0019] The RNA combination is a combination of RNA transcribed from the second gene expression cassette in the second recombinant gene engineering vector and RNA transcribed from the third gene expression cassette.

[0020] M8: Biomolecular Combination

[0021] The biomolecule combination is a combination of the fusion protein expressed in the fourth gene expression cassette in the third recombinant gene engineering vector and the sgRNA expressed in the fifth gene expression cassette, or a combination of RNA transcribed from the fourth gene expression cassette and the sgRNA expressed in the fifth gene expression cassette.

[0022] M9: Genetically engineered cells

[0023] The genetically engineered cells contain the genetic engineering vector described in M1, the recombinant genetic engineering vector described in M2, or a combination of genetic engineering vectors described in M3.

[0024] M10: Vesicle

[0025] The vesicles are those secreted by the genetically engineered cells described in M9;

[0026] M11: Transfected cells

[0027] The transfected cells are cells transfected with the vesicles described in M10;

[0028] M12: Composition

[0029] The composition comprises the genetic engineering vector described in M1, the recombinant genetic engineering vector described in M2, the combination of genetic engineering vectors described in M3, the fusion protein described in M4, the RNA described in M5, the protein combination described in M6, the RNA combination described in M7, the biomolecule combination described in M8, the genetically engineered cell described in M9, the vesicle described in M10, or the transfected cell described in M11; and

[0030] M13: Reagent Kit

[0031] The kit contains the genetic engineering vector described in M1, the recombinant genetic engineering vector described in M2, the combination of genetic engineering vectors described in M3, the fusion protein described in M4, the RNA described in M5, the protein combination described in M6, the RNA combination described in M7, the biomolecule combination described in M8, the genetically engineered cell described in M9, the vesicle described in M10, or the transfected cell described in M11.

[0032] In some implementations, the selection is made from any of the following: A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10.

[0033] A1: The amino acid sequence of the ORF3 protein of the hepatitis E virus is shown in SEQ ID NO.2;

[0034] A2: In the first recombinant gene engineering vector or the second recombinant gene engineering vector, the exogenous gene is selected from fluorescent protein gene, transcription activator protein gene, bioactive regulatory protein gene, and medicinal protein gene.

[0035] A3: In the first or second gene engineering vector, the backbone of the gene engineering vector is selected from pLVX vector, pCAGGS vector; or

[0036] In the third gene engineering vector, the backbone of the gene engineering vector is selected from pLVX vector, pCAGGS vector, and Lenti-CRISPR-V2 vector.

[0037] A4: The lentiviral vector plasmid uses pLVX as the backbone plasmid; the lentiviral packaging helper plasmid is pMD2.G; the lentiviral envelope plasmid is psPAX2;

[0038] A5: The promoters of the first gene expression cassette, the second gene expression cassette, the third gene expression cassette, the fourth gene expression cassette, or the fifth gene expression cassette are constitutive expression promoters or artificially inducible promoters;

[0039] A6: The first gene expression cassette, the second gene expression cassette, the third gene expression cassette, or the fourth gene expression cassette further contains a coding sequence for a tag peptide used to isolate and purify the protein;

[0040] The tag peptide encoding the protein for protein isolation and purification encodes a peptide segment that is directly linked to other peptide segments; or

[0041] The tag peptide used for protein isolation and purification encodes a peptide segment that also has a 1-15 amino acid linker or random amino acid sequence that does not affect the independence of domain activity between the peptide segment and other peptide segments.

[0042] A7: The hepatitis E virus ORF3 protein in the fusion protein expressed by the fourth gene expression cassette is directly linked to the Cas9 protein suitable for the CRISPR / Cas9 system; or

[0043] The ORF3 protein of the hepatitis E virus and the Cas9 protein suitable for the CRISPR / Cas9 system also have a 1-15 amino acid linker or random amino acid sequence that does not affect the independence of domain activity.

[0044] A8: In the first gene expression cassette or the fourth gene expression cassette, the ORF3 protein of the hepatitis E virus is located upstream of the fusion protein;

[0045] A9: The host cells for the genetically engineered cells are selected from HEK293T cells, HepG2 / C3A cells; and

[0046] A10: The host cells of the transfected cells are selected from HepG2 / C3A cells, S10-3 cells, TZM-bl cells, and Vero cells.

[0047] A second aspect of the present invention provides a method for preparing vesicles, wherein the method is a first method, a second method, a third method, or a fourth method;

[0048] The first method includes the following steps:

[0049] S1-1: Insert the foreign gene coding sequence into the foreign gene insertion site of the first genetic engineering vector or the second genetic engineering vector described in the first aspect of the present invention to obtain a first recombinant vector;

[0050] S1-2: Transfect vesicle-generating cells with the first recombinant vector to obtain the first recombinant cells;

[0051] S1-3: Collect the cell culture supernatant of the first recombinant cell, purify the cell culture supernatant by differential ultracentrifugation, and obtain the first vesicle;

[0052] The second method includes the following steps:

[0053] S2-1: Insert the sgDNA corresponding to the target sequence of the target gene suitable for the CRISPR / Cas9 system into the sgDNA insertion site of the third gene engineering vector to obtain the second recombinant vector;

[0054] S2-2: Transfect vesicle-generating cells with the second recombinant vector to obtain the second recombinant cells;

[0055] S2-3: Collect the cell culture supernatant of the second recombinant cell, and purify the cell culture supernatant by differential ultracentrifugation to obtain the second vesicle;

[0056] The third method includes the following steps:

[0057] S3-1: Insert the foreign gene coding sequence into the foreign gene insertion site of the first gene engineering vector or the second gene engineering vector described in the first aspect of the present invention to obtain a third recombinant vector; the backbone of the third recombinant vector is a lentiviral vector plasmid;

[0058] S3-2: Using the third recombinant vector, lentiviral packaging helper plasmid and lentiviral envelope plasmid are co-transfected into vesicle-generating cells to obtain the third recombinant cells;

[0059] S3-3: Collect the cell culture supernatant of the third recombinant cell, purify the cell culture supernatant by differential ultracentrifugation to obtain the third vesicle;

[0060] The fourth method includes the following steps:

[0061] S4-1: Insert the sgDNA corresponding to the target sequence of the target gene suitable for the CRISPR / Cas9 system into the sgDNA insertion site of the third gene engineering vector to obtain the fourth recombinant vector; the backbone of the fourth recombinant vector is a lentiviral vector plasmid;

[0062] S4-2: Using the fourth recombinant vector, lentiviral packaging helper plasmid and lentiviral envelope plasmid are co-transfected into vesicle-generating cells to obtain the fourth recombinant cells;

[0063] S4-3: Collect the cell culture supernatant of the fourth recombinant cell, purify the cell culture supernatant by differential ultracentrifugation, and obtain the fourth vesicle.

[0064] In some implementations, the following B1 and B2 are selected;

[0065] B1: The vesicle-generating cells are selected from HEK293T cells and HepG2 / C3A cells;

[0066] B2: The differential ultracentrifugation method is as follows: the first recombinant cell, the second recombinant cell, the third recombinant cell or the fourth recombinant cell are centrifuged at low speed to remove cell debris and large particles, and the supernatant is retained; then the supernatant is centrifuged again at high speed and the precipitate is retained to obtain vesicles.

[0067] A third aspect of the present invention provides a vesicle, wherein the vesicle is a first vesicle, a second vesicle, a third vesicle, or a fourth vesicle prepared by the method described in the second aspect of the present invention.

[0068] A fourth aspect of the present invention provides a method for delivering a foreign protein, wherein the method comprises transfecting a vesicle receptor cell with the first vesicle or the third vesicle of the third aspect of the present invention to deliver the foreign protein into the vesicle receptor cell.

[0069] In some implementations, the following C1 and C2 are selected;

[0070] C1: The vesicle receptor cells are selected from HEK293T cells and HepG2 / C3A cells;

[0071] C2: The exogenous protein gene is selected from fluorescent proteins, transcription activator proteins, bioactive regulatory proteins, and pharmaceutical proteins.

[0072] The fifth aspect of the present invention provides a gene editing method, wherein the method involves transfecting vesicle recipient cells with the second vesicle or the fourth vesicle, so that the fusion protein and the sgRNA edit the target gene corresponding to the sgRNA in the recipient cells.

[0073] In some implementations, the following options are selected: D1 and D2.

[0074] D1: The vesicle receptor cells are selected from HepG2 / C3A cells, S10-3 cells, TZM-bl cells, and Vero cells;

[0075] D2: The sgRNA is an sgRNA that targets the genome of herpes simplex virus.

[0076] The sixth aspect of this invention provides the use of a biological substance in the preparation of a formulation for delivering exogenous proteins, wherein the biological substance is selected from: the genetic engineering vector described in M1 of the first aspect of this invention, the recombinant genetic engineering vector described in M2, the combination of genetic engineering vectors described in M3, the fusion protein described in M4, the RNA described in M5, the protein combination described in M6, the RNA combination described in M7, the genetically engineered cell described in M9, the vesicle described in M10 or the transfected cell described in M11, or the first vesicle or the third vesicle described in the third aspect of this invention; wherein the genetic engineering vector is the first genetic engineering vector or the second genetic engineering vector; and the recombinant genetic engineering vector is the first recombinant genetic engineering vector or the second recombinant genetic engineering vector.

[0077] The first aspect of this invention provides the application of a biological substance in the preparation of a formulation for gene editing, wherein the biological substance is selected from: the gene engineering vector described in M1 of the first aspect of this invention, the recombinant gene engineering vector described in M2, the combination of gene engineering vectors described in M3, the combination of biomolecules described in M8, the gene engineering cell described in M9, the vesicle described in M10 or the transfected cell described in M11, or the second or fourth vesicle described in the third aspect of this invention; wherein the gene engineering vector is the third gene engineering vector; and the recombinant gene engineering vector is the third recombinant gene engineering vector.

[0078] Beneficial effects

[0079] This invention provides a hepatitis E virus ORF3 protein vesicle delivery system, the beneficial effects of which include:

[0080] Efficient protein expression and secretion: This system is designed independently of other viral proteins, enabling HEV ORF3 protein to be delivered to vesicles more efficiently.

[0081] Flexibility and controllability: This system does not rely on other viral proteins, giving it greater flexibility and controllability. It allows for easier operation and regulation of the vesicle delivery system to meet the needs of different research or applications.

[0082] Efficient Protein Expression in Genetic Engineering: The independence and efficiency of this vesicle delivery system make it a promising tool in the field of genetic engineering. By binding the target gene to this system, efficient protein expression and secretion can be achieved, which is of great significance for the production of important proteins, drug delivery, or vaccine development.

[0083] Targeted delivery of exogenous proteins: This system has the ability to carry exogenous proteins and can be used for targeted delivery of specific proteins, such as important proteins, drugs, or therapeutic proteins in biomedical research. This targeted delivery holds promise for applications in disease treatment, gene therapy, and other fields.

[0084] Design of a drug delivery system: This vesicle delivery system can be designed as a platform for drug delivery, in which drugs can be encapsulated within vesicles and delivered in vivo to target cells or tissues. This approach helps improve drug bioavailability, reduce toxicity, and achieve targeted drug delivery.

[0085] Gene therapy research: Combining gene vectors with this vesicle delivery system can be used for gene therapy research. This system holds promise for delivering gene materials to specific cells more precisely and efficiently to treat or correct genetic diseases.

[0086] Cellular signaling research: The delivery system described in this invention allows researchers to better understand the mechanisms of intercellular signal transduction. This is of great value for elucidating disease mechanisms, discovering new therapeutic targets, and gaining a deeper understanding of cellular biological processes.

[0087] Overall, the application of this vesicle delivery system in genetic engineering and biomedical research expands research tools and methods in areas such as protein expression, drug delivery, and gene therapy. This invention is expected to bring numerous beneficial effects to the clinical application of the HEV ORF3 protein vesicle delivery system, thereby promoting research and application development in related fields. Attached Figure Description

[0088] Figure 1 This is a diagram showing the secretion characteristics of the ORF3 protein.

[0089] Figure 2 This diagram shows the ORF3 protein vesicles entering the cell after secretion. The scale bar in Figure A indicates 200 μm.

[0090] Figure 3 This is a photograph showing the process of ORF3 protein vesicles entering cells.

[0091] Figure 4 This diagram illustrates the protein function of ORF3 protein vesicles after they carry proteins into the cell.

[0092] Figure 5 This diagram illustrates the function of ORF3 protein vesicles carrying functional proteins after they enter the cell. The scale bar in the diagram indicates 200 μm. Detailed Implementation

[0093] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0094] Example 1: Identification of the self-secretion properties of ORF3 vesicles.

[0095] I. Preparation of Recombinant Viral Vectors

[0096] pLVX-IRES-mCherry plasmid: A lentiviral vector plasmid carrying an mCherry gene expression cassette, purchased from Wuhan Miaoling Biotechnology Co., Ltd., catalog number P0424.

[0097] ORF3 protein of hepatitis E virus: referred to as ORF3 protein in this invention.

[0098] The coding sequence of ORF3 protein is as follows (SEQ ID NO.1): atgggatcaccatgtgccctagggttgttctgctgctgttcttcgtgtttctgcctatgctgcccgcgccaccggccggcc agccgtctggccgtcgtcgtgggcggcgcagcggcggtgccggcggtggtttctggggtgacagggttgattctcagcccttcgccc tcccctatattcatccaaccaaccccttcgccgccgatatcgtttcacaatccggggctggaactcgccctcggcagccgccccgcccccttggctccgcttggcgtgaccagtcccagcgcccctccgctgccccccgccgtcgatctgccccagctggggctgcgccgttga

[0099] The amino acid sequence of the ORF3 protein is as follows (SEQ ID NO.2): MGSPCALGLFCCCSSCFCLCCPRHRPASRLAVVVGGAAAVPAVVSGVTGLILSPSPSPIFIQPTPSPPISFHNPGLELALGSRPAPLAPLGVTSPSAPPLPPAVDLPQLGLRR

[0100] ORF3 plasmid: A recombinant plasmid containing the coding sequence fragment of the ORF3 protein, formed by cloning the aforementioned coding sequence of the ORF3 protein into the pCAGGS vector.

[0101] The aforementioned ORF3 plasmid was amplified by PCR using the following primers F1 (SEQ ID NO.3) and R1 (SEQ ID NO.4) to obtain amplification product 1.

[0102] Primer F1: CCCAGGACTCCTCCCTGCAGG ATGGGATCACCATGTGCCCTAG

[0103] Primer R1: TAGTCTCGAGGAATTCTCA ACGGCGCAGCCCCAG

[0104] The pLVX-IRES-mCherry plasmid was double-digested using restriction endonucleases BamHI and EcoRI to obtain a linear pLVX-IRES-mCherry plasmid. The amplification product 1 was then ligated into the linear pLVX-IRES-mCherry plasmid vector using an infusion enzyme. Thus, plasmids expressing the ORF3 protein and mCherry protein of hepatitis E virus (hereinafter referred to as pLVX-ORF3) were constructed using the pLVX-IRES-mCherry plasmid as a vector.

[0105] II. Packaging of Recombinant Lentivirals

[0106] Lentiviral packaging steps: 5.0 × 10 6 HEK293T cells were seeded in 10 cm cell culture dishes, and 10 ml of DMEM medium containing 2% fetal bovine serum was added. After 18 hours, the cell density reached approximately 80%, meeting the requirements for transfection with lentiviral plasmids. 42 µL of TransIT®-Lenti transfection reagent (Mirus, MIR 6606) and 14.0 µg of plasmid (6 µg psPAX2, 2 µg pMD2.G, 6 µg pLVX-ORF3) were mixed and incubated at room temperature for 10 minutes. The mixture was then added dropwise to the cells for co-transfection. Cells were cultured in DMEM medium containing 2% fetal bovine serum at 37°C in a 5% CO2 incubator. Cell culture supernatant was collected at 24, 48, and 72 hours post-transfection. 70 mL of cell culture supernatant was loaded into an ultracentrifuge tube, centrifuged at 20,000 g for 30 minutes to collect the precipitate, and resuspended in 0.5 mL of DMEM culture medium containing 2% fetal bovine serum to obtain lentivirus.

[0107] Lentiviral infection of target cells HepG2 / C3A: Packaged lentiviral concentrate was used to infect HepG2 / C3A cells to obtain cells stably expressing ORF3 protein. Positive cells were sorted by flow cytometry using mCherry fluorescent protein as a marker, and then cultured in SFM4 medium in a 5% CO2 cell culture incubator.

[0108] psPAX2 plasmid: purchased from Wuhan Miaoling Biotechnology Co., Ltd., catalog number P0261, is a lentiviral packaging helper plasmid carrying viral gag, pol, rev and tat genes.

[0109] pMD2.G plasmid: purchased from Wuhan Miaoling Biotechnology Co., Ltd., catalog number P0262, is a lentiviral vector plasmid, which is an envelope plasmid used for lentiviral packaging.

[0110] The pLVX (pLVX-ORF3) plasmid carries the target gene and packaging signals, providing the lentiviral genome for final insertion into the target cell genome. The psPAX2 plasmid provides viral structural proteins and enzymes (Gag / Pol / Rev / Tat) for assembling the viral core. pMD2.G provides the VSV-G envelope protein, determining the targeting and stability of viral infection. Co-transfection with all three is necessary to produce infectious lentiviral particles.

[0111] III. Detection of transfected cells

[0112] After 4 days of culture in serum-free SFM4 medium, cells were lysed with cell lysis buffer (Solarbio, R0100) and collected for Western blotting to detect β-actin housekeeping gene protein. Cell culture supernatant was collected and the following four sets of tests were performed: (1) The cell culture supernatant was treated with membrane removal agent (1% NP-40, sterile water) at 37°C for 15 min; (2) The cell culture supernatant was treated with proteinase K at 100 μg / mL, sterile water, at 37°C for 15 min; (3) The cell culture supernatant was treated with membrane removal agent (1% NP-40) at 37°C for 15 min, and then the cell culture supernatant was treated with proteinase K at 100 μg / mL at 37°C for 15 min; (4) The cell culture supernatant was not treated with membrane removal agent and proteinase K, and the cell culture supernatant was kept at 37°C for 15 min. The ORF3 protein was then detected by Western blotting to determine the secretory characteristics of ORF3 vesicles.

[0113] After 4 days of serum-free SFM4 culture, the collected supernatant was processed according to the four methods described above. Western blot analysis was performed using rabbit anti-ORF3 protein monoclonal antibody IgG (1:1000, Bioss, catalog number bs-0212R, rabbit-derived) as the primary antibody and HRP-labeled goat anti-rabbit IgG (Sigma) as the secondary antibody. Results are shown below. Figure 1 A. Collected cell lysates were analyzed by Western blot using mouse anti-β-actin monoclonal antibody IgG (Proteintech, 66009, mouse-derived) as the primary antibody and HRP-labeled goat anti-mouse IgG (Sigma) as the secondary antibody. Results are shown below. Figure 1 A.

[0114] IV. Experimental Results

[0115] See the experimental results. Figure 1Image A shows the membrane-bound secretion of ORF3 protein in HepG2 / C3A-ORF3 cells as detected by Western blotting. From top to bottom, the first image shows ORF3 secretion in the cell culture supernatant, the second shows ORF3 protein expression in cells, and the third shows the expression of the housekeeping protein β-actin in cells, reflecting the total cell count.

[0116] Therefore, the cell culture supernatant was treated with 1% NP-40 and proteinase K to degrade proteins, and then analyzed by Western blotting. The results showed that ORF3 was detectable in both the cell culture supernatant and intracellularly when neither 1% NP-40 nor proteinase K was present, proving that ORF3 protein can be secreted. When 1% NP-40 was present and proteinase K was absent, ORF3 was detectable in both the cell culture supernatant and intracellularly; when 1% NP-40 was absent but proteinase K was present, ORF3 was detectable in the cell culture supernatant; when both 1% NP-40 and proteinase K were present, ORF3 was not detectable in either the cell culture supernatant or intracellularly. NP-40 can disrupt the structure of vesicles, allowing proteinase K to digest the proteins released from the vesicles. This demonstrates that ORF3 protein can form a lipid membrane coating to protect it from degradation by proteinase K. Figure 1 A). This self-secreted lipid membrane encapsulating the ORF3 protein is named an ORF3 vesicle. The ORF3 protein can carry exogenous proteins co-expressed on the same vector (e.g., mcherry) into the vesicle, and it holds promise as a tool for preparing vesicles containing such exogenous proteins.

[0117] Example 2: ORF3 has the ability to carry exogenous proteins and secrete them outside the cell.

[0118] Plasmids expressing ORF3, EGFP, and ORF3-EGFP were constructed using pCAGGS as a vector and then transfected into HEK293T cells to express ORF3, EGFP, and ORF3-EGFP proteins. The secretion of ORF3, EGFP, and ORF3-EGFP proteins in the culture supernatant was analyzed by Western blotting.

[0119] I. Preparation of Recombinant Plasmids

[0120] pCAGGS plasmid: purchased from Wuhan Miaoling Biotechnology Co., Ltd., catalog number P0166.

[0121] pCAGGS-EGFP plasmid: The EGFP coding sequence fragment was amplified from CAGGS-EGFP-PJA1 (human) (purchased from Wuhan Miaoling Biotechnology Co., Ltd., catalog number P60717) and ligated between the multiple cloning sites EcoRI and XhoI of the pCAGGS plasmid to construct the pCAGGS-EGFP plasmid. Its biological characteristic is that it can express EGFP protein using the pCAGGS plasmid as a backbone.

[0122] pCAGGS-ORF3 plasmid: The ORF3 coding sequence fragment was amplified from the pLVX-ORF3 plasmid obtained in Example 1 and ligated between the multiple cloning sites EcoRI and XhoI of the pCAGGS plasmid to construct the pCAGGS-ORF3 plasmid. Its biological characteristics are that it uses pCAGGS as a backbone and can express the ORF3 protein.

[0123] pCAGGS-ORF3-GFP plasmid: The ORF3 coding sequence fragment was amplified from the pLVX-ORF3 plasmid obtained in Example 1 and ligated between the multiple cloning sites XhoI and NheI of the pCAGGS-EGFP plasmid to construct the coding sequence of the pCAGGS-ORF3-EGFP plasmid. Its biological characteristics are that it uses pCAGGS as a backbone and can express a fusion protein of ORF3 and EGFP proteins (the ORF3 protein coding sequence is located upstream of EGFP).

[0124] II. Transfection

[0125] HEK293T cells were seeded in 6-well plates at a density of 1.0 × 10⁻⁶. 6 Cells were added to each well at a concentration of 10% fetal bovine serum (FBS). After 18 hours, the cell density reached approximately 80%, meeting the requirements for plasmid transfection. 4 µL of jetPRIME transfection reagent (Polyplus, 101000001) and 4.0 µg of plasmid (pCAGGS-EGFP, pCAGGS-ORF3, and pCAGGS-ORF3-EGFP were mixed and incubated at room temperature for 10 minutes. Cells were then added dropwise. Cells were cultured in DMEM medium containing 2% FBS at 37°C in a 5% CO2 incubator. Cell culture supernatant and cells were collected 24 hours post-transfection.

[0126] Cell culture supernatants and cells were collected and subjected to SDS-PAGE protein electrophoresis. Rabbit anti-ORF3 protein monoclonal antibody IgG (1:1000, Bioss, catalog number bs-0212R, rabbit source) and mouse anti-GFP protein monoclonal antibody IgG (Proteintech, 66002, mouse source) were used as primary antibodies, and HRP-labeled goat anti-mouse IgG (Sigma) + HRP-labeled goat anti-rabbit IgG (Sigma) were used as secondary antibodies. Western blot analysis was performed. Results are shown below. Figure 1 B.

[0127] III. Experimental Results

[0128] Figure 1 B shows the results of detecting the secretion of the exogenous protein EGFP from ORF3 vesicles into the cell supernatant using the Western blotting method. From top to bottom, the first figure shows the secretion of each protein in the cell culture supernatant, and the second figure shows the expression of each protein in the cell.

[0129] Experimental results: After transfection of cells with plasmids expressing ORF3-EGFP protein, Western blot analysis showed that ORF3-EGFP protein was detected in both cells and cell culture supernatant. Figure 1 (B) indicates that ORF3 can carry EGFP and release it into the cell culture supernatant, demonstrating that ORF3 has the ability to carry exogenous proteins expressed in fusion into vesicles and secrete them extracellularly. The fusion expression of the target protein does not affect the ability of ORF3 to secrete into vesicles; therefore, ORF3 possesses the ability to secrete fusion proteins extracellularly and can also be used to prepare vesicles containing ORF3 fusion proteins.

[0130] Example 3: ORF3 has the ability to carry foreign proteins into cells.

[0131] I. Preparation of Recombinant Plasmids

[0132] Purchase the pCAGGS-mWasabi plasmid (Addgene, 164161), use this plasmid as a template to amplify the mWasabi fragment, and ligate it between the multiple cloning sites XhoI and NheI of the pCAGGS-ORF3 plasmid prepared in Example 2 to obtain the plasmid pCAGGS-ORF3-mWasabi. Its biological characteristics are that it uses pCAGGS as a backbone and can express the fusion protein of ORF3 protein and mWasabi protein respectively, with the ORF3 protein located upstream of the mWasabi protein.

[0133] II. Cell Transfection Experiment with Recombinant Plasmids

[0134] HepG2 / C3A cells were transfected with plasmids pCAGGS and pCAGGS-ORF3-mWasabi, respectively, in DMEM medium containing 10% fetal bovine serum for 24 h. Empty pCAGGS vector was used as a parallel negative control, and cell fluorescence was observed. Figure 2 A).

[0135] Cells and supernatants were collected and subjected to SDS-PAGE protein electrophoresis. Western blot analysis was performed on the cell culture supernatant and intracellular ORF3-mWasabi protein using rabbit anti-ORF3 protein monoclonal antibody IgG (1:1000, Bioss, catalog number bs-0212R, rabbit-derived) as the primary antibody and HRP-labeled goat anti-rabbit IgG (Sigma) as the secondary antibody. Results are shown below. Figure 2 B.

[0136] Simultaneously, cell culture supernatants transfected with pCAGGS-mWasabi plasmid were collected, and the supernatants were concentrated using exosome purification methods to obtain vesicles containing the ORF3-mWasabi fusion protein. Exosomes were purified using differential ultracentrifugation, the core principle of which is to utilize the differences in size and density between exosomes and other cellular components, separating them by progressively increasing centrifugal force. First, low-speed centrifugation (300 ×g) was used to remove live cells, followed by medium-speed centrifugation (2,000 ×g) to remove apoptotic bodies and cell debris. Finally, under ultra-high-speed centrifugation (typically ≥100,000 ×g), the exosomes were precipitated to the bottom of the tube, and the precipitate was collected to obtain vesicles.

[0137] The collected 50 μL of ORF3-mWasabi vesicles were mixed with 1.0 × 10 6 HepG2 / C3A cells or another type of liver cancer cell (S10-3 cells) were co-incubated in a cell culture incubator at 37°C and 5% CO2 in medium containing 10% fetal bovine serum. Cells were collected at 5 minutes and 60 minutes after incubation for SDS-PAGE protein electrophoresis. Western blot analysis was performed using rabbit anti-ORF3 monoclonal antibody IgG (1:1000, Bioss, catalog number bs-0212R, rabbit-derived) as the primary antibody and HRP-labeled goat anti-rabbit IgG (Sigma) as the secondary antibody. Western blot analysis was also performed using mouse anti-β-actin monoclonal antibody IgG (Proteintech, 66009, mouse-derived) as the internal control antibody and HRP-labeled goat anti-mouse IgG (Sigma) as the secondary antibody (results see [link to results]). Figure 2 C). The grayscale of the electrophoretic bands was analyzed using ImageJ software. The results are shown in [link to analysis]. Figure 2 D.

[0138] The collected 50 μL of ORF3-mWasabi vesicles (green) were mixed with 1.0 × 10⁻⁶ mol / L of urea-formyl vesicles. 6 HepG2 / C3A cells were incubated at 37°C in a 5% CO2 cell culture incubator in medium containing 10% fetal bovine serum (containing Hoechst dye labeled with a final concentration of 1.0 μg / mL for cell nuclei (blue) and Alexa Fluor® 594-WGA labeled with a final concentration of 1.0 μg / mL for cell membranes (red)). Fluorescence of HepG2 / C3A cells was observed under a fluorescence live-cell microscope, and fluorescence images were recorded at 0 min, 2 min, 4 min, 18 min, and 34 min after vesicle addition. See [Results Here] Figure 3 The fluorescence of mWasabi in the ORF3-mWasabi fusion protein is shown in green. The cell nucleus is stained with Hoechst dye at a final use concentration of 1.0 μg / mL (blue), and the cell membrane is labeled with Alexa Fluor® 594-WGA at a final use concentration of 1.0 μg / mL (red).

[0139] Figure 2 In the figures, A shows the fusion expression of ORF3 protein and green fluorescent protein mWasabi observed under a fluorescence microscope; B shows the fusion expression of ORF3 protein and green fluorescent protein mWasabi after fusion expression, and the secretion of both into cell culture cells and the supernatant using Western blotting; from top to bottom, the first figure shows the protein expression in cells, the second figure shows the secretion of protein in cell culture supernatant; C shows the detection of ORF3 protein vesicle entry into cells using Western blotting, with housekeeping protein β-actin reflecting the total cell volume; and D shows the results of analyzing the protein level of ORF3 protein vesicles entering cells using ImageJ software. Figure 3 To observe the process of ORF3 protein vesicles entering cells under a confocal microscope.

[0140] Green fluorescence was observed in the transfected cells, indicating that ORF3-mWasabi can be expressed in HepG2 / C3A cells. Figure 2 A). Western blotting was used to detect the levels of ORF3-mWasabi protein in cells and released into the cell culture supernatant. The results showed that ORF3 protein could be detected in both cells and the cell culture supernatant, further confirming that ORF3 vesicles can be expressed in cells and can carry exogenous proteins from cells to the cell culture supernatant. Figure 2B). ORF3-mWasabi vesicles secreted by HepG2 / C3A cells expressing ORF3-mWasabi were collected and co-incubated with HepG2 / C3A cells for 5 min and 60 min, respectively. Simultaneously, ORF3-mWasabi vesicles were co-incubated with S10-3 cells for 5 min and 60 min, respectively. Subsequently, the level of ORF3 protein entering the cells was detected by Western blotting. The results showed that ORF3 protein was detectable in both HepG2 / C3A and S10-3 cells after 60 min of culture, further confirming that ORF3 vesicles can carry exogenous proteins into cells and release them into the cell culture supernatant. Figure 2 C). Simultaneously, ImageJ analysis showed that the level of detectable ORF3 protein in cells after 60 min of culture was significantly higher than that after 5 min, thus confirming the process by which ORF3 vesicles carry exogenous proteins into cells. Figure 2 D). Furthermore, using Alexa Fluor® 594-WGA to label the HepG2 / C3A cell membrane, the process of ORF3-mWasabi vesicles entering the cell was observed using fluorescence microscopy. The results showed that ORF3-mWasabi gradually crossed the cell membrane over time, thus confirming that ORF3 vesicles can carry the exogenous protein mWasabi into the cell. Figure 3 Therefore, ORF3 vesicles have the potential to be developed into exogenous protein delivery systems.

[0141] Example 4: ORF3 vesicles carry exogenous proteins into cells and can perform protein functions.

[0142] I. Preparation of Recombinant Plasmids

[0143] (1) pLVX-ORF3 plasmid

[0144] For the description of the ORF3 protein and its coding sequence, please refer to Example 1.

[0145] The synthesized ORF3 coding sequence is inserted into the BamHI and EcoRI positions of the pLVX plasmid to obtain a recombinant plasmid called the ORF3 plasmid, which can express the ORF3 protein.

[0146] (2) pLVX-TAT plasmid

[0147] The TAT coding sequence is (SEQ ID NO.5): ATGGAGCCAGTAGATCCTAGACTAGAGCCCTGGAAGCATCCAGGAAGTCAGCCTAAAACTGCTTGTACCAATTGCTATTGTAAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCT TAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGACCTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAG

[0148] The synthesized TAT coding sequence was inserted into the BamHI and EcoRI positions of the pLVX plasmid, resulting in a recombinant plasmid called the TAT plasmid. This recombinant plasmid can express the TAT protein. The activity of the TAT protein is as follows: it interacts with the luc gene promoter in TZM-bl cells, activating the expression of luciferase.

[0149] (3) pLVX-ORF3-TAT plasmid

[0150] The fusion protein coding sequence, with an upstream ORF3 sequence and a downstream TAT sequence, was synthesized as follows (SEQ ID NO. 6):

[0151] ATGGGATCACCATGTGCCCTAGGGTTGTTCTGCTGCTGTTCTTCGTGTTTCTGCCTATGCTGCCCGCGCCACCGGCCGGCCAGCCGTCTGGCCGTCGTCGTGGGCGGCGCAGCGGCGGTGCCGGCGGTGGTTTCTGGGGTGACAGGGTTG ATTCTCAGCCCTTCGCCCTCCCCTATATTCATCCAACCAACCCCTTCGCCGCCGATATCGTTTCACAATCCGGGGCTGGAACTCGCCCTCGGCAGCCGCCCCGCCCCCTTGGCTCCGCTTGGCGTGACCAGTCCCAGCGCCCCTCCGCTG CCCCCCGCCGTCGATCTGCCCCAGCTGGGGCTGCGCCGTATGGAGCCAGTAGATCCTAGACTAGAGCCCTGGAAGCATCCAGGAAGTCAGCTAAAACTGCTTGTACCAATTGCTATTGTAAAAAGTGTTGCTTTCATTGCCAAGTTTGT TTCATAACAAAAGCCTTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGACCTCCTCAAGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAG

[0152] The first 339 bits are encoded as ORF3, and the last 340 bits are encoded as TAT.

[0153] The synthesized fusion protein coding sequence was inserted into the BamHI and EcoRI positions of the pLVX plasmid, and the resulting recombinant plasmid was called the ORF3-TAT plasmid. This recombinant plasmid can express a fusion protein with ORF3 protein upstream and TAT protein downstream.

[0154] (4) pLVX-Nef-TAT plasmid

[0155] The fusion protein coding sequence, with a Nef sequence upstream and a TAT sequence downstream, was synthesized, and the specific sequence is as follows (SEQ ID NO. 7):

[0156] ATGGGGGGCAGGTGGTCAAAAAGTAGCATAGTGGGATGGCCTGCTATAAGGGAAAGAATGAGGCGAGCTAGGCCAGCAGCAGAAGGGGTAGGAGCAGCGTCTCGAGATTTAGATAGACGTGGAGCAATCACAATCAATAATATAGCATCTAATAATCCTGACTCCGCCTGGCTGGAAGCACAAGAGGAAGAGGGGGATGTAGGCTTTCCAGTCAGGCCTCAGGTACCTCTAAGACCAATGACCTATAAGGGTGCTTTTGATCTCGGCTTCTTTTTAAAAGAAAAGGGGGGACTGGATGGGTTAGTTTACTCCAAGAAAAGACAAGAGATCCTTGATCTGTGGGTCTATCACACACAAGGCTTCTTCCCTGACTGGCAGTGCTACACACCAGGGCCAGGGGTGAGATATCCCCTGACCTTTGGGTGGTGCTACAAACTAGTGCCACTGGATCCAACAGAGGTAGAGGAAGCCAATAAAGGAGAGAACAACAGTCTATTACACCCCATCTGCCAGCATGGAATGGACGACGAAGACGGAGAAGTGCTGGTATGGAGATTTGACAGTAGCCTAGCACGGAGACACGTAGCCCGAGAGCTGCATCCGGAGTTCTACAAAGACTGCGGCGGTGGTGGCTCTGGTGGCGGTGGCTCCATGGAGCCAGTAGATCCTAGACTAGAGCCCTGGAAGCATCCAGGAAGTCAGCCTAAAACTGCTTGTACCAATTGCTATTGTAAAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCTTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGACCTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAG

[0157] Positions 1-621 are the Nef coding sequence, positions 622-651 are the linker coding sequence, and positions 652-912 are the TAT coding sequence. The activity of the Nef protein, as a positive control, is characterized by its extracellular secretion via exosomes.

[0158] The synthesized fusion protein coding sequence is inserted into the BamHI and EcoRI positions of the pLVX plasmid, and the resulting recombinant plasmid is called the Nef-TAT plasmid. This recombinant plasmid can express a fusion protein with Nef protein upstream and TAT protein downstream.

[0159] (5) pLVX-pX-TAT plasmid

[0160] The fusion protein coding sequence, with a pX sequence upstream and a TAT sequence downstream, was synthesized as follows (SEQ ID NO. 8):

[0161] CGTTTTGCTCGTAAGGGTGCTCTTCGTCAGAAAAACGTTATGGAGCCAGTAGATCCTAGACTAGAGCCCTGGAAGCATCCAGGAAGTCAGCCTAAAACTGCTTGTACCAATTGCTATTGTAAAAAGTGTTGCTTTCATTGCCAAGTTTGT TTCATAACAAAAGCCTTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGACCTCCTCAAGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAG

[0162] Bits 1-39 are pX encoded sequences, and bits 40-300 are TAT encoded sequences.

[0163] The activity of pX protein serves as a positive control, characterized by its extracellular secretion via exosomes.

[0164] The synthesized fusion protein coding sequence is inserted into the BamHI and EcoRI positions of the pLVX plasmid, and the resulting recombinant plasmid is called the pX-TAT plasmid. This recombinant plasmid can express a fusion protein with pX protein upstream and TAT protein downstream.

[0165] II. Preparation of Lentivirals

[0166] Lentiviral packaging steps: 5.0 × 10 6HEK293T cells were seeded in 10 cm cell culture dishes in DMEM containing 10% fetal bovine serum. After 18 hours, the cell density reached approximately 80%, meeting the requirements for transfection with lentiviral plasmids. 42 µL of TransIT®-Lenti transfection reagent (Mirus, MIR 6606), 6 µg psPAX2, 2 µg pMD2.G, and 6 µg recombinant pLVX plasmid (pLVX-ORF3, pLVX-TAT, pLVX-ORF3-TAT, pLVX-Nef-TAT, and pLVX-pX-TAT were mixed and incubated at room temperature for 10 minutes. The mixture was then added dropwise to the cells. Cells were cultured in DMEM containing 2% fetal bovine serum at 37°C in a 5% CO2 incubator. Cell culture supernatants were collected at 24, 48, and 72 hours post-transfection. 70 mL of cell culture supernatant was transferred to an ultracentrifuge tube, centrifuged at 20,000 g for 30 minutes, and the pellet was collected. The pellet was resuspended in 0.5 mL of DMEM culture medium containing 2% fetal bovine serum to obtain five lentiviruses. The five lentiviruses were named pLVX-ORF3 lentivirus, pLVX-TAT lentivirus, pLVX-ORF3-TAT lentivirus, pLVX-Nef-TAT lentivirus, and pLVX-pX-TAT lentivirus, respectively.

[0167] III. Preparation of Vesicles

[0168] Lentiviral infection of target cells HepG2 / C3A: HepG2 / C3A cells were infected with concentrated pLVX-ORF3 lentivirus to obtain cells stably expressing exogenous proteins. These cells were then cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2 cell culture incubator.

[0169] Vesicle preparation: To observe the state of HepG2 / C3A cells after lentiviral infection, the culture medium was replaced with serum-free SFM4 (20 mL). SFM4 was added to Hepes buffer at a volume ratio of 1:100. The cell culture supernatant (20 mL) was collected, and vesicles were extracted from the supernatant by centrifugation and named ORF3 vesicles. The specific experimental method was as follows: 2000g for 10 min at 4℃ to remove cell debris, and a 0.22 μm filter membrane was used to further remove large particles, retaining the supernatant. The supernatant was then centrifuged again at 100,000g for 2 h at 4℃, and the precipitate was retained. The precipitate was then resuspended in 500 μL of SFM4 to obtain ORF3 vesicles.

[0170] HepG2 / C3A cells were inoculated with the TAT lentivirus constructed above, and cultured under the same conditions as those used for preparing ORF3 vesicles to obtain vesicles, which were named TAT vesicles. The function of the vesicles carrying the TAT protein was detected using the aforementioned method.

[0171] HepG2 / C3A cells were inoculated with the aforementioned constructed ORF3-TAT lentivirus under the same culture conditions as those used for preparing ORF3 vesicles, and vesicles were obtained and named ORF3-TAT vesicles. The function of the vesicles carrying the ORF3-TAT protein was detected using the aforementioned method.

[0172] HepG2 / C3A cells were inoculated with the Nef-TAT lentivirus constructed above, and cultured under the same conditions as those used for preparing ORF3 vesicles. Vesicles were obtained and named Nef-TAT vesicles. The function of the vesicles carrying the Nef-TAT protein was detected using the aforementioned method.

[0173] HepG2 / C3A cells were inoculated with the pX-TAT lentivirus constructed above, and cultured under the same conditions as those used for preparing ORF3 vesicles. Vesicles were obtained and named pX-TAT vesicles. The function of the vesicles carrying the pX-TAT protein was detected using the aforementioned method.

[0174] Vesicle function assay: 50 μL of LORF3 vesicles were added to TZM-BL cells. TZM-BL cells were incubated at a concentration of 1.5 x 10⁻⁶ cells / cells. 5 ORF3 vesicles were added at a concentration of 100 μL / well to a 96-well plate and cultured for approximately 18 hours. When adding ORF3 vesicles, the original medium was discarded first, and then 50 μL of ORF3 vesicles were added. After incubation for 6 hours, another 50 μL of fresh culture medium was added. After incubation of ORF3 vesicles for 24 hours, ORF3 protein function was detected.

[0175] TZM-bl cells are a genetically engineered HeLa cell line. They serve as a highly sensitive sensor or reporter system for detecting and quantifying HIV infectivity. The Luciferase gene, a reporter gene, is pre-integrated into the chromosome of TZM-bl cells, with a long terminal repeat (LTR) promoter for HIV-1 at its anterior end. The LTR promoter itself has very low activity. The Tat protein produced by the virus specifically binds to the LTR, strongly trans-activating (initiating) the expression of the reporter gene downstream of the LTR. Once the reporter gene is activated, the cell synthesizes large amounts of Luciferase. A Luciferase substrate (luciferin) is added to the cells. Luciferase catalyzes a chemical reaction in the substrate, producing fluorescence. The intensity of the fluorescence is detected using a chemiluminescence analyzer (Luminometer). The fluorescence intensity is directly proportional to the amount of TAT added to the cell: a stronger fluorescence signal indicates a higher amount of TAT added to the cell.

[0176] As a control, TZM-bl cells were transformed with pLVX-ORF3, pLVX-TAT, pLVX-ORF3-TAT, pLVX-Nef-TAT, and pLVX-pX-TAT plasmids, respectively. A luciferase substrate (luciferin) was then added to the cells. Luciferase catalyzes a chemical reaction in the substrate, producing fluorescence. The intensity of the fluorescence was detected using a chemiluminescence analyzer (Luminometer).

[0177] Figure 4 In the diagram, A is an experimental schematic: ORF3-TAT vesicles carrying proteins enter TZM-BL cells, and the functional mechanism of using ORF3 to carry TAT proteins is detected. B shows the fluorescence value induced by detecting cell-secreted proteins. The first five values ​​on the horizontal axis represent the functional status of the five proteins in cells 24 hours after plasmid transfection, and the last five values ​​represent the functional status of the five proteins in cells 48 hours after plasmid transfection. C shows the fluorescence value induced by detecting the entry of vesicles obtained after cell-secreted proteins into the next cell. The first five values ​​on the horizontal axis represent the collection of secreted ORF3 vesicles, treatment of TZM-BL cells for 24 hours, and detection of the functional status of ORF3 vesicles carrying functional proteins in TZM-BL cells. The last five values ​​represent the collection of secreted ORF3 vesicles, treatment of TZM-BL cells for 48 hours, and detection of the functional status of ORF3 vesicles carrying functional proteins in TZM-BL cells. Untransfected TZM-bl cells serve as a control (Ctrl).

[0178] Experimental results are as follows Figure 4 The diagram shows the construction of the ORF3 protein, the plasmid for ORF3 protein fusion expression with a protein with a specific function, and the cell line. Figure 4 A). TZM-BL cells were transfected with plasmids to express ORF3 protein, and TZM-BL cells were transfected with plasmids to express functional TAT proteins (TAT; Nef-TAT; pX-TAT). TZM-BL cells were transfected with plasmids to express both ORF3 protein and exogenous protein (ORF3-TAT protein). After culturing the transfected cells for 24 h and 48 h, the cells were collected, and the expression of the luciferase gene initiated by TAT was evaluated by luciferase fluorescence detection. The results showed that the fluorescence values ​​of TAT protein and ORF3-TAT protein were similar, proving that the expressed ORF3 vesicle fusion functional protein plays a role in the cells, and verifying the stability of the system. Figure 4B). The culture supernatants of HepG2 / C3A cells stably expressing ORF3 protein, HepG2 / C3A cells stably expressing functional TAT protein (TAT; Nef-TAT; pX-TAT), and HepG2 / C3A cells stably expressing both ORF3 protein and the exogenous functional protein TAT were collected and incubated with TZM-BL cells. Fluorescence values ​​were detected in all cases after 24 and 48 hours of incubation, demonstrating that the TAT protein secreted by the control group without ORF3 fusion could not enter the next cell to perform its function, while the ORF3 protein vesicles carrying the functional TAT protein could enter the cell and perform its function. Figure 4 C).

[0179] ORF3 protein can be secreted to form vesicles. These vesicles, when incubated with TZM-BL cells, cannot activate the LTR to initiate Luc expression. TAT protein cannot be secreted into vesicles, but if present in TZM-BL cells, it can activate the LTR to initiate Luc expression. The ORF3-TAT fusion protein can be secreted into vesicles via ORF3 protein, and upon incubation with TZM-BL cells, it can activate the LTR to initiate Luc expression via TAT protein. Nef-TAT and pX-TAT fusion proteins can be secreted to form vesicles, thus entering TZM-BL cells and activating the LTR to initiate Luc expression via TAT protein. Fusion proteins composed of ORF3 and TAT can be secreted into vesicles. After fusion with other cells, the TAT in the fusion protein can function. Therefore, the fusion of ORF3 with functional protein peptides can guide the release of functional protein peptides in vesicle form, allowing them to enter other cells and exert their functions. ORF3 can be seen as a tool for carrying other proteins. Vectors containing the ORF3 coding sequence can be used to insert foreign genes, and then the foreign genes can be transferred into other cells via vesicles as an intermediate medium through ORF3.

[0180] Example 5: Utilizing the properties of ORF3 vesicles to exert antiviral effects.

[0181] (1) HSV-1-F strain:

[0182] The genome sequence of human herpesvirus 1 strain F can be found in GenBank: GU734771.1.

[0183] (2) HSV-1-F-eGFP strain:

[0184] The eGFP gene coding sequence was inserted upstream of the UL24 gene in the HSV-1-F strain to form a fusion expression of the eGFP and UL24 genes, resulting in a recombinant viral strain that can constitutively express green fluorescent protein eGFP.

[0185] (3) Plasmid Lenti-CRISPR-V2:

[0186] Purchased from Addgene, catalog number 52961. Contains separate Cas9 gene and gRNA expression cassettes.

[0187] (4) Insertion of sgDNA

[0188] For the HSV-1-F-eGFP strain, an sgRNA targeting the UL24 gene in the HSV genome was designed.

[0189] The target sequence of the UL24 gene in the HSV genome is: 5'-CATGCCGAGAGTGATCCCGGG-3'.

[0190] sgRNA sequence positive strand 5'-CACCGCATGCCGAGAGTGATCCCGG-3'

[0191] sgRNA sequence negative strand 5'-AAACCCGGGATCACTCTCGGCATGC-3'

[0192] Anneal the positive and negative strands of the sgRNA sequence to obtain a double strand. The linearized Lenti-CRISPR-V2 plasmid is then ligated to the double strand using DNA ligase to obtain a recombinant plasmid in which sgDNA is inserted into the gRNA scaffold.

[0193] (5) Insert ORF3

[0194] The ORF3 coding sequence was synthesized, and AgeI and BamH recognition sites were added upstream and downstream, respectively. The recombinant plasmid obtained in step (4) and the ORF3 fragment were linearized by double digestion with AgeI and BamHI. The ORF3-sgRNA sequence was fused by infusion method to construct the plasmid Lenti-CRISPR-Cas9-ORF3, which expresses ORF3-Cas9 and antiviral sgRNA. Different gene expression cassettes express Cas9-ORF3 fusion protein and sgRNA, respectively.

[0195] HEK293T cells were transfected with Lenti-CRISPR-Cas9-ORF3 in DMEM containing 10% fetal bovine serum. After 48 hours of culture, the cell culture supernatant was collected and subjected to SDS-PAGE protein electrophoresis. Western blot analysis was performed using rabbit anti-ORF3 monoclonal antibody IgG (1:1000, Bioss bs-0212R, rabbit-derived) as the primary antibody and HRP-labeled goat anti-rabbit IgG (Sigma) as the secondary antibody. Western blot analysis was also performed on the aforementioned cell culture supernatant using mouse anti-β-actin monoclonal antibody IgG (Proteintech, 66009, mouse-derived) as the internal control and HRP-labeled goat anti-mouse IgG (Sigma) as the secondary antibody. Untransfected HEK293T cells served as a parallel control. Results are shown below. Figure 5 A.

[0196] Vesicle preparation: HEK293T cell culture medium transfected with Lenti-CRISPR-Cas9-ORF3 for 24 hours was replaced with serum-free SFM4 (20 mL). SFM4 was added to Hepes buffer at a 1:100 volume ratio. The cell culture supernatant (20 mL) was collected, and vesicles were extracted from the supernatant by centrifugation and named Cas9-ORF3 vesicles. The specific experimental method was as follows: 2000g, 10 min, 4℃ to remove cell debris, further remove large particles with a 0.22 μm filter membrane, and retain the supernatant; then the supernatant was centrifuged again at 100000g, 2 h, 4℃, and the precipitate was retained. The precipitate was then resuspended with 500 μL of SFM4 to obtain Cas9-ORF3 vesicles. Cas9-ORF3 vesicle function detection: Vero cells were infected with HSV-1-F-eGFP strain and cultured in DMEM medium containing 10% fetal bovine serum at 37℃ in a 5% CO2 cell culture incubator. Vesicles prepared by adding collected cell supernatant 40 h post-infection were observed under an inverted fluorescence microscope 2 h later. Figure 5 B).

[0197] The main characteristics of vesicle-susceptible cells (cells that readily endocytose and take up vesicles) are: high membrane fluidity, a large surface area to volume ratio, and active cytoskeleton dynamics. The main cell types include: antigen-presenting cells, endothelial cells, epithelial cells with active absorptive functions, and neurons.

[0198] Figure 5In Figure 1, A shows the expression of the ORF3 and Cas9 fusion proteins detected by Western blotting, with housekeeping protein β-actin reflecting the total cell count. Figure B (left) shows cells infected with HSV-1-F-eGFP strain (containing green fluorescent protein) and incubated with protein vesicles carrying the ORF3-Cas9 fusion protein and the sgRNA of the eGFP gene in the antiviral agent, after herpes simplex virus expression was observed under fluorescence microscopy. The right image shows cells under white light. Figure C (left) shows cells in the unincubated control group observed under fluorescence microscopy, with the right image showing cells under white light.

[0199] Experimental results: Western blotting was used to detect the level of ORF3-Cas9 fusion protein in cells, and the results showed that Cas9 protein could be detected in cells. Figure 5 A) confirmed that ORF3-Cas9 can be expressed in cells. Simultaneously, observation under an inverted fluorescence microscope showed that the viral green fluorescence was reduced after the addition of ORF3-Cas9 vesicles to Vero cells (A). Figure 5 B left and Figure 5 C left), and according to Figure 5 B (Right Figure) and Figure 5 As shown in Figure C on the right, the cells in both cells are in the same state under white light, thus confirming that ORF3-Cas9 can prevent HSV-GFP from replicating in Vero cells and play an antiviral role.

[0200] Example 6: Preparation method of ORF3 vesicles.

[0201] A lentiviral plasmid pLVX-ORF3 expressing ORF3 was constructed. The viral plasmid pLVX-ORF3 and pVSV-G, psPAX2 were co-transfected into HEK293T cells, packaged to obtain lentivirus, and the lentivirus was used to infect the target cells to obtain a cell line that stably secretes ORF3 protein vesicles. The cell line was cultured, and the supernatant was collected to obtain ORF3 protein vesicles.

[0202] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A biomaterial, said biomaterial being any one of the following M1, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, M12 and M13; M1: Genetic engineering vector The genetic engineering vector is a first genetic engineering vector, a second genetic engineering vector, or a third genetic engineering vector. The first genetic engineering vector contains a first gene expression cassette, which contains the coding sequence of the ORF3 protein of hepatitis E virus and a foreign gene insertion site. After the foreign gene is inserted at the foreign gene insertion site, the fusion protein expressed by the first gene expression cassette includes the ORF3 protein of the hepatitis E virus and the protein encoded by the foreign gene. The second gene engineering vector contains a second gene expression cassette and a third gene expression cassette; the second gene expression cassette is capable of expressing the ORF3 protein gene of hepatitis E virus; the third gene expression cassette contains a foreign gene insertion site, which is used to insert and express the foreign gene. The third gene engineering vector contains a fourth gene expression cassette and a fifth gene expression cassette; the fourth gene expression cassette expresses fusion proteins including the ORF3 protein of hepatitis E virus and the Cas9 protein suitable for the CRISPR / Cas9 system; the fifth gene expression cassette contains an sgDNA insertion site corresponding to the target sequence suitable for the CRISPR / Cas9 system for targeting the target gene, and after the sgDNA is inserted into the insertion site, the fifth gene expression cassette can transcribe sgRNA for targeting the target gene; M2: Recombinant gene engineering vector The recombinant gene engineering vector is a first recombinant gene engineering vector, a second recombinant gene engineering vector, or a third recombinant gene engineering vector. The exogenous gene is inserted into the exogenous gene insertion site of the first gene engineering vector to form the first recombinant gene engineering vector. The exogenous gene is inserted into the exogenous gene insertion site of the second gene engineering vector to form the second recombinant gene engineering vector. The sgDNA is inserted into the insertion site of the third recombinant gene engineering vector to form the third recombinant gene engineering vector; M3: Genetic engineering vector combination The genetic engineering vector combination includes lentiviral vector plasmids, lentiviral packaging helper plasmids, and lentiviral envelope plasmids. The lentiviral vector plasmid is selected from the genetic engineering vector described in M1 or the recombinant genetic engineering vector described in M2. M4: Fusion protein The fusion protein is the fusion protein expressed by the first gene expression cassette in the first recombinant gene engineering vector; M5: RNA The RNA can be translated to obtain the fusion protein described in M4; M6: Protein Combination The protein combination is a combination of the protein expressed by the second gene expression cassette in the second recombinant gene engineering vector and the protein expressed by the third gene expression cassette. M7: RNA combination The RNA combination is a combination of RNA transcribed from the second gene expression cassette in the second recombinant gene engineering vector and RNA transcribed from the third gene expression cassette. M8: Biomolecular Combination The biomolecule combination is a combination of the fusion protein expressed in the fourth gene expression cassette in the third recombinant gene engineering vector and the sgRNA expressed in the fifth gene expression cassette, or a combination of RNA transcribed from the fourth gene expression cassette and the sgRNA expressed in the fifth gene expression cassette. M9: Genetically engineered cells The genetically engineered cells contain the genetic engineering vector described in M1, the recombinant genetic engineering vector described in M2, or a combination of genetic engineering vectors described in M3. M10: Vesicle The vesicles are those secreted by the genetically engineered cells described in M9; M11: Transfected cells The transfected cells are cells transfected with the vesicles described in M10; M12: Composition The composition comprises the genetic engineering vector described in M1, the recombinant genetic engineering vector described in M2, the combination of genetic engineering vectors described in M3, the fusion protein described in M4, the RNA described in M5, the protein combination described in M6, the RNA combination described in M7, the biomolecule combination described in M8, the genetically engineered cell described in M9, the vesicle described in M10, or the transfected cell described in M11; and M13: Reagent Kit The kit contains the genetic engineering vector described in M1, the recombinant genetic engineering vector described in M2, the combination of genetic engineering vectors described in M3, the fusion protein described in M4, the RNA described in M5, the protein combination described in M6, the RNA combination described in M7, the biomolecule combination described in M8, the genetically engineered cell described in M9, the vesicle described in M10, or the transfected cell described in M11.

2. The biomaterial as described in claim 1, characterized in that, Choose from any one of the following: A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10; A1: The amino acid sequence of the ORF3 protein of the hepatitis E virus is shown in SEQ ID NO.2; A2: In the first recombinant gene engineering vector or the second recombinant gene engineering vector, the exogenous gene is selected from fluorescent protein gene, transcription activator protein gene, bioactive regulatory protein gene, and medicinal protein gene. A3: In the first or second gene engineering vector, the backbone of the gene engineering vector is selected from pLVX vector, pCAGGS vector; or In the third gene engineering vector, the backbone of the gene engineering vector is selected from pLVX vector, pCAGGS vector, and Lenti-CRISPR-V2 vector. A4: The lentiviral vector plasmid uses pLVX as its backbone plasmid; The lentivirus packaging helper plasmid is pMD2.G; The lentiviral envelope plasmid psPAX2; A5: The promoters of the first gene expression cassette, the second gene expression cassette, the third gene expression cassette, the fourth gene expression cassette, or the fifth gene expression cassette are constitutive expression promoters or artificially inducible promoters; A6: The first gene expression cassette, the second gene expression cassette, the third gene expression cassette, or the fourth gene expression cassette further contains a coding sequence for a tag peptide used to isolate and purify the protein; The tag peptide encoding the protein for protein isolation and purification encodes a peptide segment that is directly linked to other peptide segments. or The tag peptide used for protein isolation and purification encodes a peptide segment that also has a 1-15 amino acid linker or random amino acid sequence that does not affect the independence of domain activity between the peptide segment and other peptide segments. A7: The hepatitis E virus ORF3 protein in the fusion protein expressed by the fourth gene expression cassette is directly linked to the Cas9 protein suitable for the CRISPR / Cas9 system; or The ORF3 protein of the hepatitis E virus and the Cas9 protein suitable for the CRISPR / Cas9 system also have a 1-15 amino acid linker or random amino acid sequence that does not affect the independence of domain activity. A8: In the first gene expression cassette or the fourth gene expression cassette, the ORF3 protein of the hepatitis E virus is located upstream of the fusion protein; A9: The host cells for the genetically engineered cells are selected from HEK293T cells, HepG2 / C3A cells; and A10: The host cells of the transfected cells are selected from HepG2 / C3A cells, S10-3 cells, TZM-bl cells, and Vero cells.

3. A method for preparing vesicles, wherein the method is a first method, a second method, a third method, or a fourth method; The first method includes the following steps: S1-1: Insert the foreign gene coding sequence into the foreign gene insertion site of the first genetic engineering vector or the second genetic engineering vector as described in claim 1 or 2 to obtain the first recombinant vector; S1-2: Transfect vesicle-generating cells with the first recombinant vector to obtain the first recombinant cells; S1-3: Collect the cell culture supernatant of the first recombinant cell, purify the cell culture supernatant by differential ultracentrifugation, and obtain the first vesicle; The second method includes the following steps: S2-1: Insert the sgDNA corresponding to the target sequence of the target gene suitable for the CRISPR / Cas9 system into the sgDNA insertion site of the third gene engineering vector to obtain the second recombinant vector; S2-2: Transfect vesicle-generating cells with the second recombinant vector to obtain the second recombinant cells; S2-3: Collect the cell culture supernatant of the second recombinant cell, and purify the cell culture supernatant by differential ultracentrifugation to obtain the second vesicle; The third method includes the following steps: S3-1: Insert the foreign gene coding sequence into the foreign gene insertion site of the first genetic engineering vector or the second genetic engineering vector as described in claim 1 or 2 to obtain a third recombinant vector; the backbone of the third recombinant vector is a lentiviral vector plasmid; S3-2: Using the third recombinant vector, lentiviral packaging helper plasmid and lentiviral envelope plasmid are co-transfected into vesicle-generating cells to obtain the third recombinant cells; S3-3: Collect the cell culture supernatant of the third recombinant cell, purify the cell culture supernatant by differential ultracentrifugation to obtain the third vesicle; The fourth method includes the following steps: S4-1: Insert the sgDNA corresponding to the target sequence of the target gene suitable for the CRISPR / Cas9 system into the sgDNA insertion site of the third gene engineering vector to obtain the fourth recombinant vector; the backbone of the fourth recombinant vector is a lentiviral vector plasmid; S4-2: Using the fourth recombinant vector, lentiviral packaging helper plasmid and lentiviral envelope plasmid are co-transfected into vesicle-generating cells to obtain the fourth recombinant cells; S4-3: Collect the cell culture supernatant of the fourth recombinant cell, purify the cell culture supernatant by differential ultracentrifugation, and obtain the fourth vesicle.

4. The preparation method according to claim 3, characterized in that, Selected from B1 and B2 below; B1: The vesicle-generating cells are selected from HEK293T cells and HepG2 / C3A cells; B2: The differential ultracentrifugation method is as follows: the first recombinant cell, the second recombinant cell, the third recombinant cell or the fourth recombinant cell are centrifuged at low speed to remove cell debris and large particles, and the supernatant is retained; then the supernatant is centrifuged again at high speed and the precipitate is retained to obtain vesicles.

5. A vesicle, said vesicle being a first vesicle, a second vesicle, a third vesicle, or a fourth vesicle prepared by the method of claims 3 and 4.

6. A method for delivering a foreign protein, the method comprising transfecting a vesicle receptor cell with the first vesicle or the third vesicle of claim 5, thereby delivering the foreign protein into the vesicle receptor cell.

7. The method as described in claim 6, characterized in that, Selected from C1 and C2 below; C1: The vesicle receptor cells are selected from HEK293T cells and HepG2 / C3A cells; C2: The exogenous protein gene is selected from fluorescent proteins, transcription activator proteins, bioactive regulatory proteins, and pharmaceutical proteins.

8. A gene editing method, wherein the method comprises transfecting vesicle recipient cells with the second vesicle or the fourth vesicle of claim 5, so that the fusion protein and the sgRNA edit the target gene corresponding to the sgRNA in the recipient cells.

9. The method as described in claim 8, characterized in that, Selected from D1 and D2 below; D1: The vesicle receptor cells are selected from HepG2 / C3A cells, S10-3 cells, TZM-bl cells, and Vero cells; D2: The sgRNA is an sgRNA that targets the genome of herpes simplex virus.

10. The use of a biological substance in the preparation of a formulation for delivering exogenous proteins, said biological substance being selected from: the genetic engineering vector described in M1 of claim 1 or 2, the recombinant genetic engineering vector described in M2, the combination of genetic engineering vectors described in M3, the fusion protein described in M4, the RNA described in M5, the protein combination described in M6, the RNA combination described in M7, the genetically engineered cell described in M9, the vesicle described in M10 or the transfected cell described in M11, or the first vesicle or the third vesicle described in claim 5; in, The genetic engineering vector is either the first genetic engineering vector or the second genetic engineering vector; The recombinant gene engineering vector is either the first recombinant gene engineering vector or the second recombinant gene engineering vector.

11. The use of a biological substance in the preparation of a formulation for gene editing, said biological substance being selected from: the gene engineering vector described in M1 of claim 1 or 2, the recombinant gene engineering vector described in M2, the combination of gene engineering vectors described in M3, the combination of biomolecules described in M8, the gene engineering cell described in M9, the vesicle described in M10 or the transfected cell described in M11, or the second or fourth vesicle described in claim 5; in, The genetic engineering vector is the third genetic engineering vector; The recombinant gene engineering vector is the third recombinant gene engineering vector.

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