An in vitro packaging method for ring virus pseudovirus particles
By assembling ring virus pseudovirus particles in HEK293T cells using eukaryotic expression plasmids, the technology gap in in vitro packaging was filled, enabling applications in viral biology research and gene therapy, and demonstrating the safe and efficient characteristics of ring virus.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHONGKE KAIPU BIOTECHNOLOGY (HUBEI) CO LTD
- Filing Date
- 2024-10-17
- Publication Date
- 2026-05-26
AI Technical Summary
Currently, there is a lack of efficient methods for obtaining pseudovirus particles of the ring virus in vitro, which limits the development of research on the biological characteristics of the virus and the application of exogenous gene fragment packaging.
Eukaryotic expression plasmids, including the full-length capsid protein gene fragment ORF1, the truncated fragment ORF1_del with an N-terminal arginine-rich region, and the gene fragment ORF1/2 covering ORF1 and ORF2, were used to express the viral capsid protein in HEK293T cells through enzyme digestion and ligation reactions, forming pseudovirus particles.
This study achieved efficient in vitro packaging of ring virus pseudovirus particles, providing a method for studying viral biological characteristics and packaging of exogenous gene fragments, filling a technological gap and demonstrating its potential as a safe and efficient viral vector.
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Figure CN119331911B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology technology, specifically to an in vitro packaging method for ring virus pseudovirus particles. Background Technology
[0002] Anelloviruses are one of the most important symbiotic virus families in the human body, belonging to the Anelloviridae family. Anelloviruses are non-enveloped viruses with a negative-sense single-stranded circular DNA genome. They are widespread in the human population and are characterized by persistent infection and high diversity.
[0003] Ring viruses have extremely high genetic diversity. Currently, more than 150 species of ring viruses have been discovered in 30 genera, among which the most abundant genera in humans are alpha (adults) and beta (children).
[0004] The ringvirus genome replicates via rolling circle replication, encoding the capsid protein (ORF1) and 5-6 other non-structural proteins. Ringviruses can be detected in a wide range of human tissues, organs, body fluids, and different cell types, indicating their extensive cell and tissue tropism.
[0005] Furthermore, numerous studies have shown that this virus is a symbiotic virus widely present in the human body, and viral infection does not cause specific diseases or bodily damage. In addition, the ring virus can maintain persistent infection in the body for a long time, and the body's immune response to it is relatively low. These characteristics indicate that this virus can serve as a safe and highly effective novel viral vector for application in gene therapy and basic research.
[0006] Pseudovirus particles are viral components that do not contain the viral genome. They are important viral building blocks for studying the biological characteristics of viruses and for use in viral vectors. Currently, there are no relevant technical methods in China for obtaining ring virus pseudovirus particles in vitro. Therefore, developing a simple and efficient method for packaging ring virus pseudovirus particles is of great value for studying the basic biological characteristics of this virus and for using these viral particles for subsequent in vitro packaging applications of exogenous gene fragments. Summary of the Invention
[0007] To address the gaps in the aforementioned technical fields, this invention provides a simple and efficient in vitro packaging method for ring virus pseudovirus particles, which can be applied to basic virology research and the development of antiviral drugs and vectors.
[0008] The aforementioned in vitro packaging method for ring virus pseudovirus particles involves eukaryotic expression of the ring virus capsid expression plasmid.
[0009] Preferably, the capsid expression plasmid is selected from at least one of the following: the full-length capsid protein gene fragment ORF1 (ORF1: 2211 bp), the truncated fragment ORF1_del (ORF1_del: 2022 bp) of the N-terminal arginine-rich region of the capsid protein gene, and the gene fragment ORF1 / 2 (ORF1 / 2: 2922 bp) covering the full-length ORF1 and ORF2 of the ring virus.
[0010] Capsid protein expression for the assembly of ring virus pseudovirus particles is independent of a specific plasmid backbone and can be carried out using a variety of plasmid vectors.
[0011] Preferably, the plasmid vector used for assembling ring virus pseudovirus particles includes the pcDNA3.1(+) eukaryotic expression vector.
[0012] Preferably, the in vitro packaging method for the ring virus pseudovirus particles includes the following steps:
[0013] S1. A capsid expression plasmid of the TTV3 genome of the alpha genus of digitovirus was synthesized by gene synthesis, and an NheI restriction site was introduced at the 5' end and a HindIII restriction site was introduced at the 3' end of the capsid expression plasmid.
[0014] S2. Select a suitable plasmid vector as the backbone plasmid, obtain the capsid expression plasmid and the sticky end product of the plasmid vector DNA by enzyme digestion, and ligate the corresponding capsid expression plasmid to the plasmid vector by T4 DNA ligase TaKaRa.
[0015] S3. The ligation product was transformed into competent cells Stable2. Positive clones were screened by ampicillin, and the plasmids of the positive clones were extracted to obtain the viral capsid protein expression plasmid: TTV3-capsid expression plasmid.
[0016] S4. Seed HEK293T cells and cultured overnight in DMEM at 37 ℃ and 5% CO2. Using lipo2000 (Thermo) transfection reagent, the TTV3-capsid expression plasmid prepared in step S3 was transfected into HEK293T cells. After transfection, the cell culture supernatant was collected.
[0017] S5. Purify the cell culture supernatant to obtain ring virus pseudovirus particles.
[0018] Preferably, the T4 ligation reaction system in step S2 is as follows: 10× ligation buffer 2ul, plasmid vector DNA 50 ng, the amount of inserted gene fragment is 65~85 ng, T4 DNA Ligase (350 U / μL) 1 μL, and sterile water is added to 20 μL.
[0019] Further preferred embodiments include: the inserted gene fragment is ORF1 with an insertion amount of 65 ng; the inserted gene fragment is ORF1_del with an insertion amount of 60 ng; and the inserted gene fragment is ORF1 / 2 with an insertion amount of 85 ng.
[0020] Preferably, the reaction temperature of the T4 ligation reaction in step S2 is 16 °C, and the ligation reaction time is 4~16 h.
[0021] Preferably, the HEK293T cell seeding density in step S4 is 2 × 10⁻⁶. 5 ~3×10 5 cell / mL.
[0022] Preferably, in step S4, the amount of TTV3-capsid expression plasmid added is 2 μg.
[0023] Preferably, in step S4, the transfection process is as follows: the three viral capsid protein expression plasmids obtained in step S3 are transfected into HEK293T cells separately. After 6-8 h of transfection, the culture medium is replaced with fresh medium and cultured for another 72 h.
[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0025] The full-length capsid protein gene fragment ORF1 of the ring virus, the truncated fragment ORF1_del (the N-terminal arginine-rich region of the capsid protein gene), and the gene fragment ORF1 / 2 covering the full-length ORF1 and ORF2 of the ring virus were expressed in eukaryotes. The ORF1 and ORF1_del gene fragments can express the full-length and truncated capsid proteins of the virus; the ORF1 / 2 gene fragment can express the viral capsid protein and related accessory proteins. These viral capsid proteins can assemble into virus-like particles in eukaryotic cells. The high diversity, persistent infectivity, low immunogenicity, and safety of the ring virus make it a safe and efficient novel viral vector for application in basic research and gene therapy. This invention constructs a simple and efficient ring virus pseudovirus particle, achieving in vitro packaging and acquisition of pseudoviruses, filling a technological gap in this field, and providing an important method for subsequent research on the biological characteristics of this virus, the in vitro packaging of exogenous genes, and vectorization applications. Attached Figure Description
[0026] Figure 1 This is a schematic diagram of the structure of TTV3-ORF1, TTV3-ORF1_del and TTV3-ORF1 / 2.
[0027] Figure 2 Electron micrographs of ring-virus particles in a cell section; among them, Figure 2 (a) Electron micrograph of ring virus particles in a cell section after transfection with a eukaryotic expression plasmid carrying the full-length ORF1 gene fragment of ring virus. Figure 2 (b) Electron micrograph of ring virus particles in cell sections after transfection with a eukaryotic expression plasmid carrying a truncated fragment of the N-terminal arginine-rich region of the ring virus, ORF1_del. Figure 2 (c) Electron micrographs of ring virus particles in cell sections after cells were transfected with a eukaryotic expression plasmid carrying the ring virus ORF1 / 2 gene fragment. Figure 2 (d) is an electron micrograph of an untransfected cell section. Detailed Implementation
[0028] The technical solution of the present invention will be further described and illustrated below through examples. All raw materials used in the examples are commercially available or prepared using conventional methods.
[0029] Example 1
[0030] An in vitro packaging method for ring virus pseudovirus particles includes the following steps:
[0031] S1. The full-length ORF1 gene fragment (ORF1: 2211 bp) of the TTV3 genome of the alpha genus of ring virus (3748 bp, GenBank number: AY666122) was synthesized by gene synthesis (Sangon Biotech (Shanghai) Co., Ltd.). An NheI restriction site was introduced at the 5' end and a HindIII restriction site was introduced at the 3' end.
[0032] S2. Select the pcDNA3.1(+) eukaryotic expression vector (5327 bp, Invitrogen V79020) as the backbone plasmid. Obtain the gene fragment ORF1 and the sticky end product of the pcDNA3.1(+) vector DNA by restriction endonuclease digestion in step S1. Ligate the corresponding gene fragment ORF1 to the pcDNA3.1(+) vector DNA using T4 DNA ligase (TaKaRa).
[0033] The ligation reaction system consisted of: 2 μL of 10× ligation buffer, 50 ng of plasmid vector DNA, 65 ng of the inserted gene fragment ORF1, 1 μL of T4 DNA Ligase (350 U / μL), and sterile water to a final volume of 20 μL.
[0034] The reaction temperature for the ligation reaction was 16 °C, and the reaction time was 4–16 h.
[0035] S3. The ligation product obtained in step S2 was transformed into competent cells Stable2 (Invitrogen, 10268-019). Positive clones were screened using ampicillin resistance, and plasmids of the positive clones were extracted to obtain cells with the following characteristics: Figure 1 The three viral capsid protein expression plasmids with the structures shown are: TTV3-ORF1 (7638 bp).
[0036] S4. In a six-hole plate, according to 2×10 5 HEK293T cells (purchased from ATCC cell bank) were seeded at a density of 1 cell / mL and cultured overnight at 37 °C and 5% CO2 in DMEM medium (containing 10% FBS, 100 U / mL penicillin and 100 µg / mL streptomycin). Using lipo2000 (Thermo) transfection reagent, 2 μg of the viral capsid protein expression plasmid TTV3-ORF1 (7638 bp) prepared in step S3 was transfected into HEK293T cells. After culturing for 72 h, the cell culture supernatant and cells were collected.
[0037] S5. Purify the cell culture supernatant to obtain the final product.
[0038] Example 2
[0039] An in vitro packaging method for ring virus pseudovirus particles includes the following steps:
[0040] S1. The fragment ORF1_del (189 bases truncated at the 5' end, ORF1_del: 2022 bp) of the N-terminal arginine-rich region of the TTV3 genome of the alpha genus of ring virus (3748 bp, GenBank number: AY666122) was synthesized by gene synthesis (Sangon Biotech (Shanghai) Co., Ltd.). An NheI restriction site was introduced at the 5' end and a HindIII restriction site was introduced at the 3' end.
[0041] S2. Select the pcDNA3.1(+) eukaryotic expression vector (5327 bp, Invitrogen V79020) as the backbone plasmid. Obtain the gene fragment ORF1_del and the sticky end product of the pcDNA3.1(+) vector DNA by restriction endonuclease digestion in step S1. Ligate the corresponding gene fragment ORF1_del to the pcDNA3.1(+) vector DNA using T4 DNA ligase (TaKaRa).
[0042] The ligation reaction system consisted of: 2 μL of 10× ligation buffer, 50 ng of plasmid vector DNA, 60 ng of the inserted gene fragment ORF1_del, 1 μL of T4 DNA Ligase (350 U / μL), and sterile water to a final volume of 20 μL.
[0043] The reaction temperature for the ligation reaction was 16 °C, and the reaction time was 4–16 h.
[0044] S3. The ligation product obtained in step S2 was transformed into competent cells Stable2 (Invitrogen, 10268-019). Positive clones were screened using ampicillin resistance, and plasmids of the positive clones were extracted to obtain cells with the following characteristics: Figure 1 The viral capsid protein expression plasmid with the structure shown is: TTV3-ORF1_del (7449 bp).
[0045] S4. In a six-hole plate, according to 2×10 5 HEK293T cells (purchased from ATCC cell bank) were seeded at a density of 1 cell / mL and cultured overnight at 37 ℃ and 5% CO2 in DMEM medium (containing 10% FBS, 100 U / mL penicillin and 100 µg / mL streptomycin). Using lipo2000 (Thermo) transfection reagent, 2 μg of the three viral capsid protein expression plasmids TTV3-ORF1_del (7449 bp) prepared in step S3 were transfected into HEK293T cells. After culturing for 72 h, the cell culture supernatant and cells were collected.
[0046] S5. Purify the cell culture supernatant to obtain the final product.
[0047] Example 3
[0048] An in vitro packaging method for ring virus pseudovirus particles includes the following steps:
[0049] S1. The ORF1 / 2 gene fragment (ORF1 / 2: 2922 bp) of the TTV3 genome (3748 bp, GenBank No.: AY666122) of the alpha genus of ring virus was synthesized by gene synthesis (Sangon Biotech, Shanghai). An NheI restriction site was introduced at the 5' end and an EcoRI restriction site was introduced at the 3' end.
[0050] S2. Select the pcDNA3.1(+) eukaryotic expression vector (5327 bp, Invitrogen V79020) as the backbone plasmid. Obtain the gene fragment ORF1 / 2 and the sticky end product of the pcDNA3.1(+) vector DNA by restriction endonuclease digestion in step S1. Ligate the corresponding gene fragment ORF1 / 2 to the pcDNA3.1(+) vector DNA using T4 DNA ligase (TaKaRa).
[0051] The ligation reaction system consisted of: 2 µL of 10× ligation buffer, 50 ng of plasmid vector DNA, 85 ng of ORF1 / 2 of the inserted gene fragment, 1 µL of T4 DNA Ligase (350 U / µL), and sterile water to a final volume of 20 µL.
[0052] The reaction temperature for the ligation reaction was 16 °C, and the reaction time was 4–16 h.
[0053] S3. The ligation product obtained in step S2 was transformed into competent cells Stable2 (Invitrogen, 10268-019). Positive clones were screened using ampicillin resistance, and plasmids of the positive clones were extracted to obtain cells with the following characteristics: Figure 1 The viral capsid protein expression plasmid with the structure shown is TTV3-ORF1 / 2 (8290 bp).
[0054] S4. In a six-hole plate, according to 2×10 5 HEK293T cells (purchased from ATCC cell bank) were seeded at a density of 1 cell / mL and cultured overnight at 37 °C and 5% CO2 in DMEM medium (containing 10% FBS, 100 U / mL penicillin and 100 µg / mL streptomycin). Using lipo2000 (Thermo) transfection reagent, 2 μg of the viral capsid protein expression plasmid TTV3-ORF1 / 2 (8290 bp) prepared in step S3 was transfected into HEK293T cells. After culturing for 72 h, the cell culture supernatant and cells were collected.
[0055] S5. Purify the cell culture supernatant to obtain the final product.
[0056] Detection and Analysis
[0057] 1. Electron microscopy observation of viral particle release process after transfection of cells with three capsid protein expression plasmids.
[0058] Cells collected from step S4 of Examples 1-3 were fixed with glutaraldehyde, then ultrathin sections were prepared and uranium-lead double staining was performed. The sections and negatively stained samples were observed under a Talos L120C electron microscope.
[0059] The results are as follows Figure 2 As shown, compared to the uninfected control, clear viral particles with a diameter of approximately 30–32 nm were observed in cell sections transfected with eukaryotic expression plasmids containing the full-length ORF1 gene fragment of the ring virus, the truncated ORF1_del fragment (N-terminal arginine-rich region), and the ORF1 / 2 gene fragment. This indicates that eukaryotic expression plasmids of the above three ring virus gene fragments can be successfully expressed in vitro and release ring virus pseudovirus particles.
[0060] It should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.
Claims
1. A method for in vitro packaging of ring virus pseudovirus particles, characterized in that, The capsid expression plasmid of the TTV3 strain of the alpha ring virus was expressed in eukaryotes, including the following steps: S1. A capsid expression plasmid of the TTV3 genome of the alpha genus of Gyrodactylus was synthesized by gene synthesis. An NheI restriction site was introduced at the 5' end of the capsid expression plasmid, and a HindIII restriction site was introduced at the 3' end. The GenBank accession number of the TTV3 genome is AY666122. The capsid expression plasmid was selected from either the full-length capsid protein gene fragment ORF1 or the truncated fragment ORF1_del of the N-terminal arginine-rich region of the capsid protein gene. The truncated fragment ORF1_del of the N-terminal arginine-rich region was obtained by truncating 189 bases from the 5' end of the full-length capsid protein gene fragment ORF1. S2. Select pcDNA3.1(+) eukaryotic expression vector as backbone plasmid, obtain capsid expression plasmid and sticky end products of plasmid vector DNA by enzyme digestion, and ligate the corresponding capsid expression plasmid to plasmid vector by T4 DNA ligase. S3. The ligation product was transformed into competent cells Stable2. Positive clones were screened by ampicillin, and the plasmids of the positive clones were extracted to obtain the viral capsid protein expression plasmid: TTV3-capsid expression plasmid. S4. Seed HEK293T cells and cultured them overnight in DMEM at 37 ℃ and 5% CO2. Using lipo2000 transfection reagent, the TTV3-capsid expression plasmid prepared in step S3 was transfected into HEK293T cells. After transfection, the cell culture supernatant was collected. S5. Purify the cell culture supernatant to obtain ring virus pseudovirus particles.
2. The method for in vitro packaging of ring virus pseudovirus particles according to claim 1, characterized in that, The T4 ligation reaction system described in step S2 is as follows: 2 μL of 10× ligation buffer, 50 ng of plasmid vector DNA, 65-85 ng of inserted gene fragment, 1 μL of T4 DNA Ligase at a concentration of 350 U / μL, and sterile water to a final volume of 20 μL.
3. The method for in vitro packaging of ring virus pseudovirus particles according to claim 2, characterized in that, The inserted gene fragment is ORF1 with an insertion amount of 65 ng; the inserted gene fragment is ORF1_del with an insertion amount of 60 ng.
4. The method for in vitro packaging of ring virus pseudovirus particles according to claim 1, characterized in that, The reaction temperature of the T4 ligation reaction in step S2 is 16 °C, and the reaction time is 4 to 16 h.
5. The method for in vitro packaging of ring virus pseudovirus particles according to claim 1, characterized in that, The seeding density of HEK293T cells in step S4 is 2 × 10⁻⁶. 5 ~3×10 5 cell / mL; in step S4, the amount of TTV3-capsid expression plasmid added is 2 μg.
6. The method for in vitro packaging of ring virus pseudovirus particles according to claim 1, characterized in that, In step S4, the specific transfection process is as follows: the viral capsid protein expression plasmid TTV3-capsid obtained in step S3 is transfected into HEK293T cells. After 6-8 h of transfection, the culture medium is replaced with fresh medium and cultured for another 72 h.
7. A ring virus pseudovirus particle, characterized in that, The ring virus pseudovirus particles are prepared by the in vitro packaging method of the ring virus pseudovirus particles according to any one of claims 1 to 6.
8. The ring virus pseudovirus particle according to claim 7, characterized in that, The application of the aforementioned ring virus pseudovirus particles in basic virology research and the development of antiviral drugs and vectors.