In-vitro packaging method of ring virus pseudovirus particles

Abstract

The invention provides an in-vitro packaging method of ring virus pseudovirus particles, and belongs to the technical field of molecular biology. According to the in-vitro packaging method of the ring virus pseudovirus particles, the capsid expression plasmid of the ring virus is subjected to eukaryotic expression. The finger ring virus pseudovirion can be used as a safe and efficient novel virus vector to be applied to the fields of gene therapy and fundamental research, and the technical method provided by the invention can promote fundamental research of the finger ring virus and application research of related vectorization.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular belongs to an in vitro packaging method of anellovirus pseudovirus particles. Background Art

[0002] Anellovirus is the most important symbiotic virus family in the body, belonging to the Anelloviridae family. Anellovirus is a non-enveloped virus with a negative-sense single-stranded circular DNA genome. It is widely prevalent in the human population and has the characteristics of persistent infection and high diversity.

[0003] Anelloviruses have extremely high genetic diversity. Currently, 30 genera and more than 150 species of anellovirus have been discovered, of which the most abundant in humans are the alpha genera (adults) and beta genera (children).

[0004] The anellovirus genome replicates via a rolling circle replication mechanism and encodes the capsid protein (ORF1) and 5-6 other non-structural proteins. Anelloviruses can be detected in a variety of human tissues, organs, body fluids, and different cell types, indicating their broad cell and tissue tropism.

[0005] In addition, a large number of studies have shown that the virus is a symbiotic virus that is widely present in the human body, and viral infection does not cause specific diseases or damage to the body. In addition, the ring virus can maintain a long-term continuous infection in the body, and the body's immune response level to it is low. The above characteristics indicate that the virus can be used as a safe and efficient new type of viral vector for gene therapy and basic research.

[0006] Pseudoviral particles are viral components that do not contain viral genomes. They are important viral components for studying the biological characteristics of viruses and for use in viral vectors. At present, there are no relevant technical methods for obtaining pseudoviral particles of anellovirus in vitro in China. Therefore, developing a simple and efficient method for packaging pseudoviral particles of anellovirus is of great value for studying the basic biological characteristics of the virus and using the viral particles for subsequent in vitro packaging of exogenous gene fragments. Summary of the invention

[0007] In view of the gap in the above technical field, the present invention provides a simple and efficient in vitro packaging method for anellovirus pseudoviral particles, which can be applied to basic virology research and the development of antiviral drugs and vectors.

[0008] The in vitro packaging method of anellovirus pseudovirus particles is to perform eukaryotic expression on the capsid expression plasmid of anellovirus.

[0009] Preferably, the capsid expression plasmid is at least selected from the full-length capsid protein gene fragment ORF1 (ORF1: 2211 bp), the N-terminal arginine-rich region truncated fragment ORF1_del (ORF1_del: 2022 bp) of the capsid protein gene, and the gene fragment ORF1 / 2 (ORF1 / 2: 2922 bp) covering the full-length ORF1 and ORF2 of the anellovirus.

[0010] The expression of capsid proteins used for the assembly of anellovirus pseudoviral particles does not rely on a specific plasmid backbone and can be carried out via a variety of plasmid vectors.

[0011] Preferably, the plasmid vector used for assembling anellovirus pseudoviral particles includes the pcDNA3.1(+) eukaryotic expression vector.

[0012] Preferably, the in vitro packaging method of anellovirus pseudoviral particles comprises the following steps: S1. synthesizing the capsid expression plasmid of the anellovirus alpha genus TTV3 genome by gene synthesis, and introducing an NheI restriction site at the 5' end and a HindIII restriction site at the 3' end of the capsid expression plasmid; 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 connect the corresponding capsid expression plasmid with the plasmid vector by T4 DNA ligase TaKaRa; S3. The ligation product was transformed into competent cells Stable2, and positive clones were screened by ampicillin. The positive clone plasmids were extracted to obtain the viral capsid protein expression plasmid: TTV3-capsid expression plasmid; S4. Inoculate HEK293T cells and culture in DMEM at 37°C and 5% CO 2 The cells were cultured overnight under the same conditions, and the TTV3-capsid expression plasmids prepared in step S3 were transfected into HEK293T cells using lipo2000 (Thermo) transfection reagent. After the transfection, the cell culture supernatant was collected. S5. Purify the cell culture supernatant to obtain anellovirus pseudovirus particles.

[0013] Preferably, the T4 ligation reaction system described in step S2 is: 10×ligation buffer 2ul, plasmid vector DNA 50ng, the amount of inserted gene fragment is 65-85ng, T4 DNA Ligase (350 U / μL) 1 μL, and sterile water is added to 20 μL.

[0014] Further preferably, the inserted gene fragment is ORF1, and the inserted amount is 65 ng; the inserted gene fragment is ORF1_del, and the inserted amount is 60 ng; the inserted gene fragment is ORF1 / 2, and the inserted amount is 85 ng.

[0015] Preferably, the reaction temperature of the T4 ligation reaction in step S2 is 16° C., and the ligation reaction time is 4 to 16 hours.

[0016] Preferably, the HEK293T cell seeding density in step S4 is 2×10 5 ~3×10 5 cell / mL.

[0017] Preferably, in step S4, the amount of TTV3-capsid expression plasmid added is 2 μg.

[0018] Preferably, in step S4, the specific process of transfection is to transfect the three viral capsid protein expression plasmids prepared in step S3 into HEK293T cells separately, replace the culture medium with fresh medium 6 to 8 hours after transfection, and continue culturing for 72 hours.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The full-length capsid protein gene fragment ORF1 of the anellovirus, the truncated fragment ORF1_del of 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 anellovirus are eukaryotically expressed. The ORF1 and ORF1_del gene fragments can express the full-length capsid protein and truncated capsid protein of the virus; the ORF1 / 2 gene fragment can express the viral capsid protein and related auxiliary proteins. The above-mentioned viral capsid proteins can be assembled into virus-like particles in eukaryotic cells. The high diversity, persistent infectivity, low immunogenicity and safety of the anellovirus make it a safe and efficient new type of viral vector, which can be applied to basic research and gene therapy. The present invention constructs a simple and efficient anellovirus pseudovirus particle, realizes the packaging and acquisition of pseudovirus in vitro, fills the technical gap in this field, and provides an important method for subsequent research on the biological characteristics of the virus, the in vitro packaging of exogenous genes and the vector application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the structure of TTV3-ORF1, TTV3-ORF1_del and TTV3-ORF1 / 2.

[0021] Figure 2 This is an electron micrograph of anellovirus particles in a cell section; Figure 2(a) is an electron microscopic image of anellovirus particles in cell sections after cells were transfected with a eukaryotic expression plasmid carrying the full-length ORF1 gene fragment of anellovirus. Figure 2 (b) is an electron microscopic image of anellovirus particles in cell sections after cells were transfected with a eukaryotic expression plasmid carrying the truncated fragment ORF1_del of the N-terminal arginine-rich region of the anellovirus. Figure 2 (c) is an electron microscopic image of anellovirus particles in cell sections after cells were transfected with a eukaryotic expression plasmid carrying anellovirus ORF1 / 2 gene fragment. Figure 2 (d) Electron micrograph of a cell section without transfection. DETAILED DESCRIPTION

[0022] The technical scheme of the present invention is further described below by way of examples. The raw materials used in the examples can be purchased from the market or prepared by conventional methods.

[0023] Example 1 An in vitro packaging method for anellovirus pseudoviral particles comprises the following steps: S1. The full-length ORF1 gene fragment (ORF1: 2211 bp) of the anellovirus alpha genus TTV3 genome (3748 bp, GenBank No.: AY666122) was synthesized by gene synthesis (Sangon Biotech (Shanghai) Co., Ltd.), and an NheI restriction site was introduced at the 5' end and a HindIII restriction site was introduced at the 3' end; S2. Select pcDNA3.1(+) eukaryotic expression vector (5327 bp, Invitrogen V79020) as the backbone plasmid, obtain the gene fragment ORF1 and the sticky end product of pcDNA3.1(+) vector DNA by restriction endonuclease digestion in step S1, and connect the corresponding gene fragment ORF1 with pcDNA3.1(+) vector DNA by T4 DNA ligase (TaKaRa); The ligation reaction system was as follows: 10×ligation buffer 2 μL, plasmid vector DNA 50 ng, insert gene fragment ORF1 65 ng, T4 DNA Ligase (350 U / μL) 1 μL, and sterile water to 20 μL; The reaction temperature of the ligation reaction is 16 °C, and the ligation reaction time is 4 to 16 h; S3. The ligation product obtained in step S2 was used to transform competent cells Stable2 (Invitrogen, 10268-019), and positive clones were screened by ampicillin resistance. The plasmids of positive clones were extracted to obtain the following Figure 1Three viral capsid protein expression plasmids of the indicated structures: TTV3-ORF1 (7638 bp); S4, in a six-well plate at 2×10 5 HEK293T cells (purchased from ATCC cell bank) were inoculated at a density of 100 cells / mL and cultured overnight at 37°C, 5% CO2 using DMEM medium (containing 10% FBS, 100 U / mL penicillin and 100 µg / mL streptomycin). 2 μg of the viral capsid protein expression plasmid TTV3-ORF1 (7638 bp) prepared in step S3 was transfected into HEK293T cells using lipo2000 (Thermo) transfection reagent. The cell culture supernatant and cells were collected after culturing for 72 h. S5. Purify the cell culture supernatant to obtain.

[0024] Example 2 An in vitro packaging method for anellovirus pseudoviral particles comprises the following steps: S1. The truncated fragment ORF1_del (5' end truncated by 189 bases, ORF1_del: 2022 bp) of the N-terminal arginine-rich region of the anellovirus alpha genus TTV3 genome (3748 bp, GenBank No.: AY666122) was synthesized by gene synthesis (Sangon Biotech (Shanghai) Co., Ltd.), and an NheI restriction site was introduced at the 5' end and a HindIII restriction site was introduced at the 3' end; S2. Select 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 pcDNA3.1(+) vector DNA by restriction endonuclease digestion in step S1, and connect the corresponding gene fragment ORF1_del with pcDNA3.1(+) vector DNA by T4 DNA ligase (TaKaRa); The ligation reaction system was as follows: 10×ligation buffer 2 μL, plasmid vector DNA 50 ng, insert gene fragment ORF1_del 60 ng, T4 DNA Ligase (350 U / μL) 1 μL, and sterile water was added to 20 μL; The reaction temperature of the ligation reaction is 16 °C, and the ligation reaction time is 4 to 16 h; S3. The ligation product obtained in step S2 was used to transform competent cells Stable2 (Invitrogen, 10268-019), and positive clones were screened by ampicillin resistance. The plasmids of positive clones were extracted to obtain the following Figure 1 Virus capsid protein expression plasmids of the indicated constructs: TTV3-ORF1_del (7449 bp); S4, in a six-well plate at 2×10 5 HEK293T cells (purchased from ATCC Cell Bank) were inoculated at a density of 10 cells / mL and cultured in DMEM medium (containing 10% FBS, 100 U / mL penicillin and 100 µg / mL streptomycin) at 37°C and 5% CO 2 Under the conditions, the cells were cultured overnight, and 2 μg of the three viral capsid protein expression plasmids TTV3-ORF1_del (7449 bp) prepared in step S3 were transfected into HEK293T cells using lipo2000 (Thermo) transfection reagent. The cells were cultured for 72 h and the cell culture supernatant and cells were collected; S5. Purify the cell culture supernatant to obtain.

[0025] Example 3 An in vitro packaging method for anellovirus pseudoviral particles comprises the following steps: S1. The ORF1 / 2 gene fragment (ORF1 / 2: 2922 bp) of the anellovirus alpha genus TTV3 genome (3748 bp, GenBank No.: AY666122) was synthesized by gene synthesis (Sangon Biotech (Shanghai) Co., Ltd.), and an NheI restriction site was introduced at the 5' end and an EcoRI restriction site was introduced at the 3' end; S2, select 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 pcDNA3.1(+) vector DNA by restriction endonuclease digestion in step S1, and connect the corresponding gene fragment ORF1 / 2 with pcDNA3.1(+) vector DNA by T4 DNA ligase (TaKaRa); The ligation reaction system was as follows: 10×ligation buffer 2 µL, plasmid vector DNA 50 ng, insert gene fragment ORF1 / 2 85 ng, T4 DNA Ligase (350 U / µL) 1 µL, and sterile water to 20 µL; The reaction temperature of the ligation reaction is 16 °C, and the ligation reaction time is 4 to 16 h; S3. The ligation product obtained in step S2 was used to transform competent cells Stable2 (Invitrogen, 10268-019), and positive clones were screened by ampicillin resistance. The plasmids of positive clones were extracted to obtain the following Figure 1 Virus capsid protein expression plasmids of the indicated constructs: TTV3-ORF1 / 2 (8290 bp); S4, in a six-well plate at 2×10 5 HEK293T cells (purchased from ATCC Cell Bank) were inoculated at a density of 10 cells / mL and cultured in DMEM medium (containing 10% FBS, 100 U / mL penicillin and 100 µg / mL streptomycin) at 37°C and 5% CO 2 Cultured overnight under the conditions, 2 μg of the viral capsid protein expression plasmid TTV3-ORF1 / 2 (8290 bp) prepared in step S3 was transfected into HEK293T cells using lipo2000 (Thermo) transfection reagent, and cultured for 72 h to collect the cell culture supernatant and cells; S5. Purify the cell culture supernatant to obtain.

[0026] Detection and analysis 1. Electron microscopic observation of the release of viral particles after transfection of cells with three capsid protein expression plasmids The cells in step S4 of Examples 1 to 3 were collected and fixed with glutaraldehyde, then ultrathinly sliced ​​and stained with uranium and lead; the slices and negatively stained samples were observed with a Talos L120C electron microscope.

[0027] The results are as follows Figure 2 As shown, compared with the uninfected control, clear virus particles with a diameter of about 30-32 nm can be observed in the cell sections transfected with the eukaryotic expression plasmids of the full-length ORF1 gene fragment of the anellovirus, the truncated fragment ORF1_del of the N-terminal arginine-rich region, and the ORF1 / 2 gene fragment. This indicates that the eukaryotic expression plasmids of the gene fragments of the above three anelloviruses can be successfully expressed in vitro and release anellovirus pseudovirions.

[0028] It should be understood that the above embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. An in vitro packaging method for anellovirus pseudoviral particles, characterized in that: The capsid expression plasmid of anellovirus was expressed in eukaryotic cells.

2. The in vitro packaging method of anellovirus pseudoviral particles according to claim 1, characterized in that: The capsid expression plasmid is at least selected from the full-length capsid protein gene fragment ORF1, the N-terminal arginine-rich region truncated fragment ORF1_del of the capsid protein gene, and the gene fragment ORF1 / 2 covering the full-length ORF1 and ORF2 of the anellovirus; the plasmid vector used for assembling anellovirus pseudoviral particles includes the pcDNA3.1(+) eukaryotic expression vector.

3. The in vitro packaging method of anellovirus pseudoviral particles according to claim 1, characterized in that: The in vitro packaging method of anellovirus pseudoviral particles comprises the following steps: S1. synthesizing the capsid expression plasmid of the anellovirus alpha genus TTV3 genome by gene synthesis, and introducing an NheI restriction site at the 5' end and a HindIII restriction site at the 3' end of the capsid expression plasmid; 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 connect the corresponding capsid expression plasmid with the plasmid vector by T4 DNA ligase TaKaRa; S3. The ligation product was transformed into competent cells Stable2, and positive clones were screened by ampicillin. The positive clone plasmids were extracted to obtain the viral capsid protein expression plasmid: TTV3-capsid expression plasmid; S4, inoculating HEK293T cells, using DMEM culture medium to culture overnight at 37°C, 5% CO2, using lipo2000Thermo transfection reagent, transfecting the TTV3-capsid expression plasmid prepared in step S3 into HEK293T cells, and collecting the cell culture supernatant after the transfection; S5. Purify the cell culture supernatant to obtain anellovirus pseudovirus particles.

4. The in vitro packaging method of anellovirus pseudoviral particles according to claim 3, characterized in that: The T4 ligation reaction system described in step S2 is: 10×ligation buffer 2ul, plasmid vector DNA 50ng, the amount of inserted gene fragment is 65~85ng, the amount of T4 DNA Ligase with a concentration of 350U / μL is 1μL, and sterile water is added to 20μL.

5. The in vitro packaging method of anellovirus pseudoviral particles according to claim 4, characterized in that: The inserted gene fragment is ORF1, and the inserted amount is 65 ng; the inserted gene fragment is ORF1_del, and the inserted amount is 60 ng; the inserted gene fragment is ORF1 / 2, and the inserted amount is 85 ng.

6. The in vitro packaging method of anellovirus pseudoviral particles according to claim 3, characterized in that: The reaction temperature of the T4 ligation reaction described in step S2 is 16° C., and the ligation reaction time is 4 to 16 h.

7. The in vitro packaging method of anellovirus pseudoviral particles according to claim 3, characterized in that: The seeding density of HEK293T cells described in step S4 was 2 × 10 5 ~3×10 5 cell / mL; in step S4, the amount of TTV3-capsid expression plasmid added was 2 μg.

8. The in vitro packaging method of anellovirus pseudoviral particles according to claim 3, characterized in that: In step S4, the specific process of transfection is to transfect the viral capsid protein expression plasmid TTV3-capsid expression plasmid prepared in step S3 into HEK293T cells respectively, replace the fresh culture medium after 6-8 hours of transfection, and continue to culture for 72 hours.

9. An anellovirus pseudoviral particle, characterized in that The anellovirus pseudovirion particles are prepared by the in vitro packaging method of the anellovirus pseudovirion particles according to any one of claims 1 to 8.

10. The anellovirus pseudovirion according to claim 9, characterized in that The anellovirus pseudovirus particles are used in basic virology research and the development of antiviral drugs and vectors.

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