Delivery system and application

Abstract

The invention discloses a delivery system and application, and belongs to the technical field of biomedicine. The delivery system provided by the invention solves the problem of lack of an efficient non-toxic delivery system at present. The delivery system is an ORF3 protein vesicle encoded by hepatitis E virus (HEV), and the preparation method of the delivery system comprises the following steps: constructing a lentiviral plasmid pLVX-ORF3 for expressing ORF3, co-transfecting the virus plasmid pLVX-ORF3, pVSV-G and psPAX2 to an HEK293T cell, packaging to obtain a lentivirus, infecting a target cell with the lentivirus to obtain a cell line for secreting the ORF3 protein vesicle, and carrying out freeze drying on the cell line to obtain the ORF3 protein vesicle secreting the ORF3 protein vesicle. And culturing the cell line secreting the ORF3 protein vesicles, and collecting supernatant to obtain the ORF3 protein vesicles. The delivery system can independently deliver vesicles, has the capability of carrying foreign proteins, and can be applied to the fields of biomedical research, drug development, protein engineering and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a delivery system and application thereof. Background Art

[0002] The current delivery system is mainly a lipid particle delivery system, which has problems such as limited delivery volume and high price. In order to solve these technical problems, it is urgent to develop a new delivery system. ORF3 protein is a multifunctional small protein encoded by the Hepatitis E Virus (HEV). Previous studies have suggested that ORF3 protein can package viral particles into vesicles and mediate the release of vesicle-encapsulated viral particles, and vesicle-encapsulated viral particles have the ability to infect different types of cells. Therefore, exploring the function of ORF3 protein in carrying exogenous protein secretion will help provide a new path for the development and application of new vesicle delivery systems. Summary of the invention

[0003] The invention discloses a delivery system and a preparation method and application thereof, which solve the technical problems of the current delivery system, namely, limited delivery amount and high price.

[0004] The technical solution of the present invention is as follows: Application of ORF3 protein vesicles encoded by hepatitis E virus in the preparation of delivery systems.

[0005] The ORF3 protein vesicle is prepared by constructing a lentiviral plasmid pLVX-ORF3 expressing ORF3, co-transfecting the viral plasmid pLVX-ORF3, pVSV-G and psPAX2 into HEK293T cells, packaging to obtain a lentivirus, infecting target cells with the lentivirus to obtain a cell line secreting ORF3 protein vesicles, culturing the cell line secreting ORF3 protein vesicles, collecting the supernatant, and obtaining the ORF3 protein vesicle.

[0006] The delivery system is used in the targeted delivery of exogenous proteins.

[0007] The application of the delivery system in preparing a drug delivery platform.

[0008] The application of the delivery system in gene therapy drugs.

[0009] Beneficial Effects The present invention provides a hepatitis E virus ORF3 protein vesicle delivery system, which has the following beneficial effects: Efficient protein expression and secretion: The system is designed to be independent of other viral proteins, allowing HEV ORF3 protein to be delivered more efficiently in vesicles. This helps to increase the expression level of the target protein and promote its secretion outside the cell.

[0010] Flexibility and controllability: The system does not rely on other viral proteins and has greater flexibility and controllability. The vesicle delivery system can be more conveniently operated and regulated to meet the needs of different research or applications.

[0011] Efficient protein expression in genetic engineering: The independence and efficiency of this vesicle delivery system make it an important tool in the field of genetic engineering. By combining the target gene with this system, efficient protein expression and secretion can be achieved, which is of great significance for the production of important proteins, drug loading or vaccine preparation.

[0012] Targeted delivery of exogenous proteins: The 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 can be used in disease treatment, gene therapy and other fields.

[0013] Design of drug delivery system: The vesicle delivery system can be designed as a drug delivery platform, where the drug can be encapsulated in the vesicle and delivered to the target cells or tissues in vivo through the system. This approach helps to improve the bioavailability of the drug, reduce toxicity, and achieve targeted drug delivery.

[0014] Gene therapy research: Combining gene vectors with this vesicle delivery system can be used for gene therapy research. This system helps to deliver genetic materials to specific cells more accurately and efficiently to treat or correct genetic diseases.

[0015] Cell signaling research: Through the delivery system described in the present invention, researchers can better understand the signal transduction mechanism between cells. This is of great value in revealing disease mechanisms, discovering new therapeutic targets, and gaining a deeper understanding of cell biological processes.

[0016] In general, the application of this vesicle delivery system in the field of genetic engineering and biomedical research has expanded the research tools and methods in protein expression, drug delivery and gene therapy. This invention is expected to bring many beneficial effects to the clinical application of the vesicle delivery system of HEV ORF3 protein, thereby promoting the research and application development in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Figure 1 is a graph showing the secretion characteristics of ORF3 protein, wherein A is a graph showing the secretory ORF3 protein of HepG2 / C3A-ORF3 cells wrapped in a capsule when detected by immunoblotting, wherein from top to bottom, the first graph shows the secretion of ORF3 in the cell culture supernatant, the second graph shows the expression of ORF3 protein in cells, and the third graph shows the expression of housekeeping protein β-actin in cells, reflecting the total amount of cells, and B is a graph showing the secretion of ORF3 vesicles carrying exogenous protein GFP from cells to the cell supernatant when detected by immunoblotting, wherein from top to bottom, the first graph shows the secretion of each protein in the cell culture supernatant, and the second graph shows the expression of each protein in cells; Figure 2 Figure 1 is a diagram of ORF3 protein vesicles entering cells after secretion, wherein A is the fusion expression of ORF3 protein and green fluorescent protein mWasabi observed under a fluorescence microscope, B is the fusion expression of ORF3 protein and green fluorescent protein mWasabi in cells detected by immunoblotting and secreted into the cell culture supernatant, wherein from top to bottom, the first figure is the protein expression in cells, the second figure is the protein secretion in the cell culture supernatant, C is the detection of ORF3 protein vesicles entering cells by immunoblotting, housekeeping protein β-actin reflects the total amount of cells, D is the result of analyzing the protein level of ORF3 protein vesicles entering cells by ImageJ software, and E is the process of ORF3 protein vesicles entering cells observed under a confocal microscope; Figure 3 This is a diagram for detecting the protein function of ORF3 protein vesicles after the protein is carried into the cell, wherein A is a schematic diagram of ORF3 vesicles carrying proteins into TZM-BL cells and detecting the function of the carried proteins, B is the fluorescence value excited by detecting cell secreted proteins, the first 5 values ​​on the horizontal axis in the figure are the detection of the function of the ORF3 vesicle fusion functional protein expressed in the cell 24 hours after the plasmid is transfected, and the last 5 values ​​are the detection of the function of the ORF3 vesicle fusion functional protein expressed in the cell 48 hours after the plasmid is transfected, C is the fluorescence value excited by detecting the secretion of cell secretory proteins into the next cell, the first 6 values ​​on the horizontal axis in the figure are the collection of secreted ORF3 vesicles, the treatment of TZM-BL cells for 24 hours, the detection of the function of ORF3 vesicles carrying functional proteins into TZM-BL cells, the last 6 values ​​are the collection of secreted ORF3 vesicles, the treatment of TZM-BL cells for 48 hours, the detection of the function of ORF3 vesicles carrying functional proteins into TZM-BL cells; Figure 4To detect the function of ORF3 protein vesicles carrying functional proteins into cells, A is the expression of ORF3 protein and functional protein Cas9 protein and antiviral sgRNA fusion detected by immunoblotting, housekeeping protein β-actin reflects the total amount of cells, B on the left is the cell image of the expression of herpes virus after cells were infected with herpes simplex virus with green fluorescent protein (HSV-GFP) and incubated with ORF3 protein vesicles carrying effector protein Cas9 protein and antiviral sgRNA, and the right is the cell image under white light. Figure C on the left is the cell image of the expression of herpes virus in the control group without incubation observed by fluorescence microscopy, and the right is the cell image under white light. DETAILED DESCRIPTION

[0018] Example 1 Identification of the self-secretory nature of ORF3 vesicles.

[0019] (1) Experimental methods: pLVX-IRES-mCherry was used as a vector to construct a plasmid expressing ORF3 protein fused with mCherry (hereinafter referred to as pLVX-ORF3), which was transfected with packaging plasmids psPAX2 and pMD2, packaged into lentivirus, and infected into target cells HepG2 / C3A to obtain cells stably expressing ORF3 protein. Positive cells were sorted by flow cytometry and then cultured. After 4 days of serum-free culture, the following four groups of tests were performed: treatment with membrane remover (1% NP-40) at 37°C for 15 min; digestion with 100 μg / mL Proteinase K at 37°C for 15 min; treatment with membrane remover (1% NP-40) at 37°C for 15 min, followed by digestion with 100 μg / mL Proteinase K at 37°C for 15 min; no treatment with membrane remover or Proteinase K, followed by Western blotting to detect ORF3 protein to determine the secretory properties of ORF3 vesicles.

[0020] (2) Experimental results: The cell culture supernatant was treated with a combination of membrane remover 1% NP-40 and proteinase K to degrade proteins, and then analyzed by Western blotting. The results showed that when neither 1% NP-40 nor proteinase K was present, ORF3 could be detected in the cell culture supernatant and cells, proving that ORF3 protein can be secreted. When 1% NP-40 was present and proteinase K was absent, ORF3 could be detected in the cell culture supernatant and cells; when 1% NP-40 was absent and proteinase K was present, ORF3 could be detected in the cell culture supernatant; when 1% NP-40 and proteinase K were present, ORF3 could not be detected in the cell culture supernatant or cells, thus proving that ORF3 protein can form a lipid membrane coating to protect ORF3 protein from degradation by proteinase K ( Figure 1 A). We named this self-secreted lipid membrane-encapsulated ORF3 protein ORF3 vesicle.

[0021] Example 2 ORF3 has the ability to carry foreign proteins and secrete them outside the cell.

[0022] (1) Experimental method: pCAGGS was used as a vector to construct plasmids expressing ORF3, GFP, and ORF3-GFP. The plasmids were then transfected into HEK293T cells to express ORF3, GFP, and ORF3-GFP proteins. The secretion of ORF3, GFP, and ORF3-GFP proteins in the culture supernatant was analyzed by Western blotting.

[0023] (2) Experimental results: After the cells were transfected with the plasmid expressing ORF3-GFP protein, ORF3-GFP protein was detected in the cells and in the cell culture supernatant by Western blot analysis ( Figure 1 B) shows that ORF3 can carry GFP and release it into the cell culture supernatant. ORF3 has the ability to carry exogenous proteins and secrete them outside the cell.

[0024] Example 3 ORF3 has the ability to carry foreign proteins into cells.

[0025] (1) Experimental method: Construct a pCAGGS plasmid for fusion expression of ORF3 and mWasabi. Transfect HepG2 / C3A cells with the plasmid pCAGGS-ORF3-mWasabi with a flexible peptide linker and culture for 24 hours. Then collect the cell culture supernatant and concentrate the cell culture supernatant by exosome purification to obtain ORF3-mWasabi vesicles. The collected ORF3-mWasabi vesicles were co-incubated with HepG2 / C3A or S10-3 cells and immediately observed under a fluorescent live cell microscope.

[0026] (2) Experimental results: Green fluorescence was observed in the transfected cells, indicating that ORF3-mWasabi can be expressed in the 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 both in cells and in the cell culture supernatant, further confirming that ORF3 vesicles can be expressed in cells and can carry exogenous proteins and be released from cells into the cell culture supernatant ( Figure 2 B). After the ORF3-mWasabi vesicles secreted by HepG2 / C3A cells expressing ORF3-mWasabi were collected, the ORF3-mWasabi vesicles were co-incubated with HepG2 / C3A cells for 5 minutes and 60 minutes, respectively, and the ORF3-mWasabi vesicles were co-incubated with S10-3 cells for 5 minutes and 60 minutes, respectively. Subsequently, the level of ORF3 protein entering the cells was detected by Western blotting. The results showed that ORF3 protein could be detected in both HepG2 / C3A and S10-3 cells after 60 minutes of culture, further confirming that ORF3 vesicles can carry exogenous proteins into cells and release them into the cell culture supernatant ( Figure 2 C). At the same time, analysis by ImageJ showed that the level of ORF3 protein detected in cells after 60 minutes of culture was significantly higher than that after 5 minutes of culture, confirming that ORF3 vesicles carry exogenous proteins into cells ( Figure 2 D). Furthermore, the membrane fluorescent probe DiD was used to label the HepG2 / C3A cell membrane, and the process of ORF3-mWasabi vesicles entering the cell was observed under a fluorescence microscope. The results showed that over time, ORF3-mWasabi gradually passed through the cell membrane, confirming that ORF3 vesicles can carry the exogenous protein mWasabi into the cell ( Figure 2 E).

[0027] Example 4 ORF3 vesicles carry exogenous proteins into cells and can exert protein functions.

[0028] (1) Experimental methods: pLVX vector was used to construct HepG2 / C3A cells stably expressing ORF3 protein; HepG2 / C3A cells stably expressing functional TAT protein were constructed as negative controls, including HepG2 / C3A cells expressing TAT protein, Nef-TAT protein and pX-TAT protein; HepG2 / C3A cells stably expressing ORF3 protein fused with exogenous functional protein to express ORF3-TAT protein were constructed, and all cells were cultured to T75 with a cell seeding density of 4X10^6. The state of HepG2 / C3A cells stably expressing ORF3 protein fused with exogenous functional protein to express ORF3-TAT protein was observed, and the culture medium was replaced with serum-free SFM4 (20 mL). SFM4 was added with Hepes buffer at a ratio of 1:100. The supernatant (20 mL) was collected, and then the ORF3 vesicles in the supernatant were extracted by centrifugation: the specific experimental method was to remove cell debris at 2000g, 10min, 4℃, and further remove large particles with a 0.22μm filter membrane, and retain the supernatant; the supernatant was then centrifuged again at 100000g, 2h, 4℃, and the precipitate was retained, and then the precipitate was resuspended with 500μL SFM4 to obtain ORF3 vesicles; an equal amount of 50μLORF3 vesicles was added to TZM-BL cells. TZM-BL cells were added to a 96-well plate at 1.5X10^5 cells / mL and 100μL / well for about 18h. When adding ORF3 vesicles, the original liquid was first discarded, and then 50μL ORF3 vesicles were added. After incubation for 6h, 50μL fresh culture medium was added. After incubation of ORF3 vesicles for 24h, protein function was detected.

[0029] (2) Experimental results: Construction of plasmids expressing ORF3 protein and ORF3 protein fusion with proteins with specific functions Schematic diagram of cell lines ( Figure 3 A). TZM-BL cells were transfected with plasmids expressing ORF3 protein, TZM-BL cells were transfected with plasmids expressing functional TAT protein (TAT / Nef-TAT / pX-TAT), and TZM-BL cells were transfected with plasmids expressing ORF3 protein and exogenous protein (ORF3-TAT protein). The transfected cells were cultured for 24h and 48h, respectively, and the supernatant was collected. The expression of luciferase gene initiated by TAT was evaluated by luc fluorescence detection. The results showed that the fluorescence values ​​of TAT protein and ORF3-TAT protein were similar, proving that the expression of ORF3 vesicle fusion functional protein played a role in the cell and verified the stability of the system ( Figure 3B). 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 ORF3 protein and exogenous functional protein TAT were collected and incubated with TZM-BL cells respectively. After 24h and 48h of incubation, fluorescence values ​​were detected, proving that ORF3 protein vesicles carrying TAT functional protein can enter cells and play a role ( Figure 3 C).

[0030] Example 5 Utilize the ORF3 vesicle properties to exert antiviral effects.

[0031] (1) Experimental method: Construct Lenti-CRISPR plasmid Lenti-CRISPR-Cas9-ORF3, which expresses ORF3 fusion with Cas9 and antiviral sgRNA. HEK293T cells were transfected with Lenti-CRISPR-Cas9-ORF3. After 48 hours, the cell culture supernatant was collected and detected by Western blotting ( Figure 4 A). Vero cells were infected with HSV-GFP virus, and the collected cell supernatant was added for culture 40 hours after infection. After 2 hours, the cells were observed under an inverted fluorescence microscope ( Figure 4 B).

[0032] (2) Experimental results: Western blotting was used to detect the level of ORF3-Cas9 protein in cells. The results showed Cas9 protein can be detected in cells ( Figure 4 A) confirmed that ORF3-Cas9 can be expressed in cells. At the same time, after adding ORF3-Cas9 vesicles to Vero cells, the observed virus green fluorescence weakened ( Figure 4 B Left and Figure 4 C left), and according to Figure 4 B right picture and Figure 4 The right figure C shows that the cell states of the two are the same under white light, thus confirming that ORF3-Cas9 can prevent HSV-GFP from replicating in Vero cells and play an antiviral role.

[0033] Example 6. Method for preparing ORF3 vesicles.

[0034] A lentiviral plasmid pLVX-ORF3 expressing ORF3 was constructed, and the viral plasmid pLVX-ORF3 and pVSV-G, psPAX2 were co-transfected into HEK293T cells to package the lentivirus, and the lentivirus was used to infect target cells to obtain a cell line that stably secreted ORF3 protein vesicles, and the cell line was cultured and the supernatant was collected to obtain ORF3 protein vesicles.

Claims

1. Application of ORF3 protein vesicles encoded by hepatitis E virus in the preparation of delivery systems.

2. The use according to claim 1, characterized in that: The ORF3 protein vesicle is prepared by constructing a lentiviral plasmid pLVX-ORF3 expressing ORF3, co-transfecting the viral plasmid pLVX-ORF3, pVSV-G and psPAX2 into HEK293T cells, packaging to obtain a lentivirus, infecting target cells with the lentivirus to obtain a cell line secreting ORF3 protein vesicles, culturing the cell line secreting ORF3 protein vesicles, collecting the supernatant, and obtaining the ORF3 protein vesicle.

3. Use of the delivery system according to claim 1 in the targeted delivery of exogenous proteins.

4. Use of the delivery system according to claim 1 in preparing a drug delivery platform.

5. Use of the delivery system according to claim 1 in gene therapy drugs.

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