Construction method of finger ring virus infectious clone

Abstract

The invention provides a construction method of a finger ring virus infectious clone, and belongs to the technical field of gene engineering. The construction method of the finger ring virus infectious clone comprises the following steps: respectively inserting two alpha finger ring virus full-length genomes into a skeleton plasmid according to an end-to-end sequence to obtain an infectious clone ANEalpha 3, and then transfecting to obtain finger ring virus particles. The finger ring virus 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 genetic engineering, and in particular to a method for constructing an anellovirus infectious clone. 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 anelloviruses have been discovered, of which the most abundant in humans are the alpha genera (adults) and beta genera (children). The anellovirus genome replicates through a rolling circle replication mechanism and encodes the capsid protein (ORF1) and 5 to 6 other non-structural proteins.

[0004] Anelloviruses can be detected in a variety of human tissues, organs, body fluids, and different cell types, indicating their wide range of cell and tissue tropism. In addition, a large number of studies have shown that anelloviruses are symbiotic viruses that are widely present in the human body, and viral infection does not cause specific diseases or damage to the body. In addition, anelloviruses can maintain long-term continuous infection in the body, and the body's immune response level to them is low. The above characteristics indicate that anelloviruses can be used as a safe and efficient new type of viral vector for application in gene therapy and basic research.

[0005] At present, there are no cell and animal models for anellovirus infection in China, nor any technical methods for isolating and culturing the virus. Infectious cloning is a key technical means for isolating and culturing viruses in vitro. Therefore, developing a simple and efficient anellovirus infectious cloning technology method is of great value for isolating and culturing the disease, conducting subsequent basic virology research, and packaging foreign genes in vitro and transforming them into viral vectors. Summary of the invention

[0006] In view of the gap in the above technical field, the present invention provides a simple and efficient method for constructing anellovirus infectious clones of anellovirus.

[0007] The method for constructing anellovirus infectious clone comprises the following steps: The full-length genomes of two anelloviruses of the alpha genus (3748 bp, GenBank No.: AY666122, TTV3) were inserted into the backbone plasmid in a head-to-tail order to obtain the infectious clone ANE_alpha3.

[0008] The infectious clone construction method does not rely on a specific plasmid backbone and can be carried out through a variety of plasmid vectors. Preferably, the backbone plasmid includes a pUC57 cloning vector (2710 bp).

[0009] Preferably, the full-length genome of TTV3 (3748 bp, GenBank No.: AY666122) is obtained by gene synthesis, and HindIII restriction sites are added to the 5' end and the 3' end, respectively.

[0010] Preferably, the full-length gene fragment of TTV3 is inserted into the backbone plasmid in a single restriction digestion manner through the HindIII restriction site to obtain a plasmid containing a TTV single-copy genome; the plasmid containing the TTV single-copy genome is digested through the HindIII restriction site, and the full-length gene fragment of TTV3 (3748 bp, (GenBank No.: AY666122) is recovered by 1% agarose gel.

[0011] Preferably, the ANE_alpha3 plasmid is obtained by homologous recombination.

[0012] Preferably, the plasmid containing the TTV single copy genome is amplified by PCR using primers 5'-CTTGAAATTTTAATGAAAGCCAAGTTTGAAGCTT-3' and 5'-GCATGCAGGCCTCTGC-3' to obtain a linearized plasmid DNA containing the TTV single copy genome.

[0013] Preferably, the full-length gene fragment (3748 bp) of TTV3 is amplified by PCR using primers 5'-CAGAGGCCTGCATGCAAGCTTCAAACTTGGCTTTCATTAAAATTTCAAG-3' and 5'-CCAAGTTTGAAGCTTACACGAAATTGGTCTTGGGCTT-3' to obtain the homologous recombinant gene fragment TTV3_R (3778 bp).

[0014] Preferably, the linearized plasmid DNA containing a single copy of the TTV genome and TTV3_R (3778 bp) are homologously recombined and ligated using the ClonExpress® II One Step Cloning Kit, and the infectious clone plasmid ANE_alpha3 is obtained by positive clone screening and plasmid extraction.

[0015] The ANE_alpha3 plasmid contains two copies of the anellovirus genome, which are combined in a head-to-tail manner; the sequence order of a single viral genome is not fixed and can be initiated from multiple genomic sites. The clone drives the expression of virus-related proteins through the virus's own promoter and assembles into infectious mature virus particles.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The high diversity, persistent infectivity, low immunogenicity and safety of Anellovirus make it a safe and efficient new type of viral vector for use in gene therapy and basic research. However, there is no technical method for isolating and culturing the virus in China. The present invention constructs a simple and efficient viral infectious clone to achieve in vitro viral packaging and acquisition, filling the technical gap in this field and providing an important method for subsequent in vitro viral packaging and vector transformation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the ANE_alpha3 plasmid structure.

[0018] Figure 2 This is an electron micrograph of anellovirus particles in the cell culture supernatant after cells were transfected with ANE_alpha3.

[0019] Figure 3 This is an electron micrograph of anellovirus particles in cell sections after cells were transfected with ANE_alpha3. DETAILED DESCRIPTION

[0020] 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.

[0021] Example 1 The method for constructing an anellovirus infectious clone comprises the following steps: First, the full-length genome of TTV3 (3748 bp, GenBank No.: AY666122) was obtained by gene synthesis, and HindIII restriction sites were added at the 5' and 3' ends respectively; pUC57 cloning vector (2710 bp) was selected as the backbone plasmid, and the full-length gene fragment of TTV3 was inserted into the pUC57 cloning vector by single restriction digestion (HindIII restriction site) to obtain pUC57_TTV3 plasmid (6458 bp); ANE_alpha3 plasmid (10,206 bp) was obtained by homologous recombination as follows: pUC57_TTV3 plasmid was digested by HindIII, and the full-length gene fragment of TTV3 (3748 bp) was recovered by 1% agarose gel; primers 5'- pUC57_TTV3 plasmid was amplified by PCR using 5'-CAGAGGCCTGCATGCAAGCTTCAAACTTGGCTTTCATTAAAATTTCAAG-3' and 5'-CCAAGTTTGAAGCTTACACGAAATTGGTCTTGGGCTT-3' to obtain the linearized pUC57_TTV3 plasmid DNA. The full-length gene fragment of TTV3 (3748 bp) was amplified by PCR using 5'-CAGAGGCCTGCATGCAAGCTTCAAACTTGGCTTTCATTAAAATTTCAAG-3' and 5'-CCAAGTTTGAAGCTTACACGAAATTGGTCTTGGGCTT-3' to obtain the homologous recombinant gene fragment TTV3_R (3778 bp). The linearized pUC57_TTV3 DNA (6458 bp) and TTV3_R (3778 bp) were ligated by homologous recombination, and the infectious clone plasmid ANE_alpha3 (10,206 bp) was obtained through positive clone screening and plasmid extraction.

[0022] Example 2 Transfection of cells with infectious clones and observation of virus particles by electron microscopy Inoculate HEK293T cells at a density of 2*10 5cell / mL, DMEM medium (containing 10% FBS, 100 U / mL penicillin and 100 µg / mL streptomycin) was cultured overnight at 37 °C and 5% CO2. The next day, 2 μg ANE_alpha3 plasmid was transfected into HEK293T cells using lipo2000 (Thermo) transfection reagent, and the cell culture supernatant and cells were collected after continued culture for 72 h. The cell culture supernatant was filtered through a 0.22 μm filter, 4 mL of the culture supernatant was taken, diluted to 10 mL with PBS, and purified by filtering using EXODUS H-600 (20 nm filter membrane), and finally 150 μL of PBS was eluted, and 50 μL of negative staining was taken for sample preparation; the cells were fixed with glutaraldehyde and ultrathin sections were prepared; the sections and negative staining samples were observed by Talos L120C electron microscope.

[0023] The results are as follows Figure 2 and Figure 3 As shown, compared with the uninfected control group Figure 2 (a) and Figure 3 (a) The culture supernatant of cells transfected with ANE_alpha3 plasmid in the experimental group Figure 2 (b) and cell sections Figure 3 Clear virus particles can be observed in (b), with a diameter of about 30~32 nm, indicating that ANE_alpha3 can be used as an infectious clone to successfully obtain anellovirus particles.

[0024] 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. A method for constructing an infectious clone of anellovirus, characterized in that: The full-length genomes of two anelloviruses of the alpha genus were inserted into the backbone plasmid in a head-to-tail order to obtain the infectious clone ANE_alpha3.

2. The method for constructing anellovirus infectious clone according to strong claim 1, characterized in that: The backbone plasmid includes pUC57 cloning vector.

3. The method for constructing anellovirus infectious clone according to strong claim 1, characterized in that: The full-length genome of TTV3 was obtained by gene synthesis, and HindIII restriction sites were added to the 5' and 3' ends respectively.

4. The method for constructing an anellovirus infectious clone according to strong claim 1, characterized in that: The full-length gene fragment of TTV3 was inserted into the backbone plasmid by single restriction digestion to obtain a plasmid containing a single-copy genome of TTV; the plasmid containing a single-copy genome of TTV was digested by HindIII restriction site, and the full-length gene fragment of TTV3 was recovered by 1% agarose gel.

5. The method for constructing anellovirus infectious clone according to strong claim 1, characterized in that: The ANE_alpha3 plasmid was obtained by homologous recombination.

6. The method for constructing anellovirus infectious clone according to strong claim 1, characterized in that: The backbone plasmid_TTV3 plasmid was PCR amplified using primers 5'-CTTGAAATTTTAATGAAAGCCAAGTTTGAAGCTT-3' and 5'-GCATGCAGGCCTCTGC-3' to obtain linearized plasmid DNA containing a single copy of the TTV genome.

7. The method for constructing anellovirus infectious clone according to strong claim 1, characterized in that: The full-length gene fragment (3748 bp) of TTV3 was amplified by PCR using primers 5'-CAGAGGCCTGCATGCAAGCTTCAAACTTGGCTTTCATTAAAATTTCAAG-3' and 5'-CCAAGTTTGAAGCTTACACGAAATTGGTCTTGGGCTT-3' to obtain the homologous recombinant gene fragment TTV3R.

8. The method for constructing anellovirus infectious clone according to strong claim 1, characterized in that: The linearized plasmid DNA containing a single copy of TTV genome was ligated with TTV3R by homologous recombination using ClonExpress® II One Step Cloning Kit, and the infectious clone plasmid ANE_alpha3 was obtained by positive clone screening and plasmid extraction.

9. An infectious clonal plasmid ANE_alpha3, characterized in that The infectious clone plasmid ANE_alpha3 is prepared by the construction method of the anellovirus infectious clone described in any one of claims 1 to 8.

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