A nucleotide sequence for inducing RNA interference and reducing and eliminating viral contamination in cells and its application

Abstract

The present invention discloses a dsRNA sequence and combination that can be used to reduce and eliminate Sf-Rhabdovirus contamination in Sf cells, as well as corresponding siRNA sequences, and DNA sequences and combinations corresponding to the dsRNA and combination, as well as the corresponding siRNA sequences. The present invention also discloses a method for reducing and eliminating Sf-Rhabdovirus in Sf cells using the above-mentioned nucleic acid sequence, and an Sf-Rhabdovirus-free cell line obtained thereby.

Description

Technical Field

[0001] The present invention relates to a nucleotide sequence and combination for inducing RNA interference and reducing and eliminating viral contamination in cells, a method for obtaining a virus-free cell line using this group of nucleotide sequences and combinations, and a continuous cell line free of contaminating viruses obtained using this method, wherein the virus-free cell line is derived from an organism or cell population contaminated by a virus. Background Art

[0002] The Sf9 cell-based baculovirus expression system has been widely used for the expression and production of vaccines, proteins, and genes. However, in 2014, a new type of rhabdovirus was discovered in this cell line, posing a potential safety risk for its use as a source of clinical therapeutic drugs.

[0003] Spodoptera frugiperda rhabdovirus (Sf-rhabdovirus) is a novel rhabdovirus found in Sf (Spodoptera frugiperda) cells (e.g., Sf9, Sf21). The virus was discovered by researchers at the U.S. Food and Drug Administration (FDA) using degenerate PCR and massively parallel sequencing (MPS). The Sf-rhabdovirus genome is 13,584 base pairs long and primarily consists of five genes encoding conserved proteins: N (nucleocapsid), P (phoprotein), M (matrix), G (glycoprotein), and L (ploymerase). An additional, unidentified gene encodes a non-conserved protein, X, whose function is unknown. Sf-rhabdovirus is a single-stranded (-)RNA virus. Viral replication involves RNA-dependent RNA polymerase within the virion, which transcribes RNA into mRNA, which in turn produces the corresponding proteins. The virus primarily reproduces in the cytoplasm, where it is released by budding. It cannot reproduce alone and can only multiply within living cells.

[0004] There are two main strategies for controlling Sf-Rhabdovirus: one is to remove it during sample production and purification; the other is to remove it directly from Sf9 cells. Removing Sf-Rhabdovirus during the production and purification process increases production costs as drug production increases. Furthermore, the continued presence of Sf-Rhabdovirus in the source cells creates a potential contamination risk, inevitably impacting production. Directly removing Sf-Rhabdovirus from Sf9 cells, on the other hand, avoids viral contamination at the source and effectively controls Sf-Rhabdovirus.

[0005] New insect cell lines that do not contain exogenous viruses have been isolated and used as improved research tools and safer biomanufacturing platforms. In order to completely remove the Sf-rhabdovirus from Sf9 cells, Griekobeck used nucleoside drugs (e.g., 6-azauridine) to treat a starting culture consisting of several cells, and through further amplification and culture, obtained a cell line without Sf-rhabdovirus (patent application number CN201680071306.3). This patent application attempted a method of co-inhibition with multiple drugs in actual screening. The application of multiple antiviral drugs will affect cell metabolism and biosynthesis processes, aggravate cell damage, and affect the ability of cells to recombine and produce viruses after screening. Although other studies have shown that without the addition of antiviral drugs, cell lines with similar growth characteristics and better vitality were obtained through screening (patent application number CN201910317758.0), the efficiency of obtaining virus-free cell lines by this method is very low.

[0006] In summary, the current method of inhibiting Sf-rhabdovirus in Sf9 cells mainly uses nucleoside chemical drugs, but their inhibition mode is broad-spectrum antiviral. While inhibiting the virus, it may hinder the synthesis and transcription of the cell's own genes, causing certain damage to the cells.

[0007] Therefore, there is still a need in the art for methods that can effectively and stably obtain and establish virus-free cell lines from virus-contaminated organisms or cell populations, as well as cell lines that do not contain contaminating viruses and can maintain their original cell biological characteristics and functions (for example, cell viability, ability to produce recombinant viruses, etc.).

[0008] RNA interference (RNAi) is a gene silencing phenomenon induced by double-stranded RNA (dsRNA). When double-stranded RNA homologous to the coding region of endogenous mRNA is introduced into cells, it causes the mRNA to degrade, thereby inducing gene silencing. RNAi was first discovered in Caenorhabditis elegans in 1998 and has since been found in eukaryotic organisms such as fungi, fruit flies, Arabidopsis thaliana, insects, and mammals. The main mechanism of RNAi is that a small single-stranded RNA consisting of 20-30 nucleotides forms a complex with the Argonaute protein. This complex then targets the target RNA sequence through the Watson-Crick base pairing principle, cleaving the corresponding RNA and causing gene silencing.

[0009] Three types of small interfering RNA exist in animal cells: siRNAs (small interfering RNAs), miRNAs (microRNAs), and piRNAs (PIWI-interacting RNAs), each with distinct production mechanisms and effector functions. In insect cells, the RNase III enzyme Dicer-2 recognizes double-stranded RNA (dsRNA) in the cytoplasm and cleaves it into 21nt siRNAs. One strand binds to the Ago2 protein to form the RNA-induced silencing complex (RISC), while the complementary strand is degraded. The function of Dicer-2 and the RISC complex requires the participation of multiple cofactors, including the dsRNA-binding protein Loqs-PD, Ars2, and heat shock proteins. These proteins assist in the biogenesis of siRNAs by stabilizing the RNA-protein complex or by promoting conformational changes. Following formation of the RNA silencing complex, the 3' end of the RNA becomes methylated, ultimately allowing the siRNA-loaded RISC to mature and recognize the target RNA for cleavage based on base pairing.

[0010] Currently, RNAi has been widely used in fields such as antiviral, gene regulation, and gene therapy. There are three main types of RNA interference: (1) chemically synthesized siRNA, (2) in vitro transcription of long-chain dsRNA and specific or nonspecific siRNA, and (3) siRNA expression vectors. The siRNA expression vector inserts the DNA template sequence corresponding to the siRNA into an expression plasmid. After the plasmid is transferred into the cell, RNA transcription is initiated, forming a hairpin structure of shRNA (small hairpin RNA), which is then cleaved into siRNA under the action of the Dicer enzyme.

[0011] The chemical synthesis method is to customize short siRNA molecules, each targeting a single target. Commercial companies mainly design and customize them for mammalian cells. Currently, commercial siRNA expression vectors are also targeted for in vivo expression in mammalian cells. For Sf9 cells, it is necessary to design and construct plasmids by yourself, and each plasmid targets a single target. A lot of preliminary research is required to determine the effective plasmid, which takes a long time. Therefore, it is necessary to design appropriate siRNA to solve these problems. Long-chain dsRNA is cut into multiple small siRNA molecules after being transferred into cells. These siRNAs can act on multiple targets, thereby improving the efficiency of RNA interference.

[0012] The present invention utilizes RNA interference (RNAi), specifically long-stranded dsRNA, to inhibit Sf-Rhabdovirus replication. By isolating and culturing single-cell clones or multi-cell clones, an Sf-Rhabdovirus-free cell line (SF-RVN) is generated. The RNAi approach not only avoids the addition of exogenous drugs but also reduces risks. Furthermore, there are currently no reports of using RNAi to inhibit Sf-Rhabdovirus in Sf9 cells. Summary of the Invention

[0013] The present invention provides a nucleic acid molecule or a combination thereof for inducing RNA interference and reducing and eliminating viral contamination in cells, including deoxyribonucleic acid (RNA) or a combination thereof and ribonucleic acid (DNA) or a combination thereof.

[0014] The present invention provides a dsRNA, the sequence of which is any one of SEQ ID NOs: 1-8 or a combination thereof.

[0015] In a specific embodiment of the present invention, the dsRNA sequence is: a sequence composed of SEQ ID NO: 1 and SEQ ID NO: 3; a sequence composed of SEQ ID NO: 2 and SEQ ID NO: 3; a sequence composed of SEQ ID NO: 3 and SEQ ID NO: 4; a sequence composed of SEQ ID NO: 3 and SEQ ID NO: 8; a sequence composed of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 8; or a sequence composed of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0016] In certain embodiments, the dsRNA is a modified dsRNA obtained by modifying the nucleotides in the above sequence.

[0017] The present invention also provides a dsRNA combination, which is composed of any two or more of the above-mentioned dsRNAs.

[0018] In a specific embodiment of the present invention, the dsRNA combination is: a combination of dsRNAs shown in SEQ ID NO: 1 and SEQ ID NO: 3; a combination of dsRNAs shown in SEQ ID NO: 2 and SEQ ID NO: 3; a combination of dsRNAs shown in SEQ ID NO: 3 and SEQ ID NO: 4; a combination of dsRNAs shown in SEQ ID NO: 3 and SEQ ID NO: 8; a combination of dsRNAs shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 8; or a combination of dsRNAs shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0019] The present invention also provides siRNA molecules obtained by cleaving the above dsRNA or dsRNA combination.

[0020] In another embodiment of the present invention, the siRNA may consist of any 14-30 consecutive nucleotides within any one of the sequences shown in SEQ ID NOs: 1-8 or a combination thereof, preferably 16-25 consecutive nucleotides, more preferably 18-21 consecutive nucleotides.

[0021] The present invention also provides a DNA sequence encoding the above dsRNA. In a specific embodiment of the present invention, the DNA sequence encoding the above dsRNA is any one of SEQ ID NOs: 9-16 or a combination thereof.

[0022] In certain specific embodiments of the present invention, the DNA sequence encoding the above-mentioned dsRNA is: a sequence combined by SEQ ID NO: 9 and SEQ ID NO: 11; a sequence combined by SEQ ID NO: 10 and SEQ ID NO: 11; a sequence combined by SEQ ID NO: 11 and SEQ ID NO: 12; a sequence combined by SEQ ID NO: 11 and SEQ ID NO: 16; a sequence combined by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 16; or a sequence combined by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0023] In certain embodiments, the DNA is a modified DNA obtained by modifying the nucleotides of the above sequence.

[0024] The present invention also provides a DNA combination encoding a dsRNA combination, wherein the DNA combination consists of any two or more of the above SEQ ID NOs: 9-16.

[0025] In certain specific embodiments of the present invention, the DNA combination encoding the dsRNA combination is: a combination of DNAs shown in SEQ ID NO: 9 and SEQ ID NO: 11; a combination of DNAs shown in SEQ ID NO: 10 and SEQ ID NO: 11; a combination of DNAs shown in SEQ ID NO: 11 and SEQ ID NO: 12; a combination of DNAs shown in SEQ ID NO: 11 and SEQ ID NO: 16; a combination of DNAs shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 16; or a combination of DNAs shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0026] In certain embodiments of the present invention, a DNA encoding the above-mentioned siRNA is provided.

[0027] The present invention also provides a delivery vector comprising the above-mentioned nucleic acid molecules (including RNA or DNA) or a combination thereof. The delivery vector is selected from a viral vector and a non-viral vector. Preferably, the non-viral vector is selected from the group consisting of liposomes, plasmid vectors, and phage vectors. Preferably, the viral vector is selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, and hybrid viral vectors. More preferably, the viral vector is selected from an adeno-associated viral vector. The purpose of the delivery vector is to deliver the above-mentioned nucleic acid molecules into cells to induce RNA interference.

[0028] The present invention also provides a kit comprising the RNA (including dsRNA, siRNA, etc.) or a combination thereof, DNA or a combination thereof, and a delivery vector according to the present invention.

[0029] On the other hand, the present invention also provides a method for establishing and obtaining a virus-free cell line, using the above-mentioned nucleic acid molecules (including RNA or DNA) or a combination thereof, or a delivery vector containing the above-mentioned nucleic acid molecules (including RNA or DNA) or a combination thereof, to reduce and eliminate viral contamination in cells through RNA interference technology.

[0030] The specific method is as follows:

[0031] (1) Obtaining cells from virus-contaminated organisms or cell populations;

[0032] (2) after washing the cells, introducing the nucleic acid molecule or combination thereof or a delivery vector comprising the nucleic acid molecule or combination thereof into the obtained cells to induce RNA interference;

[0033] (3) washing and resuspending the cells, and separating the cells into single cells or multiple cells;

[0034] (4) The isolated single cell or multiple cells are cultured and expanded in a culture medium suitable for cell growth and division to produce single-cell clones or multi-cell clones, thereby obtaining a virus-free cell line.

[0035] In certain embodiments of the present invention, the method for establishing and obtaining a virus-free cell line further comprises removing a portion from the cell clone or the culture medium to detect the presence of the virus.

[0036] In one aspect, the virus contaminating the organism or cell population described herein is an Sf rhabdovirus. In another aspect, the virus-contaminated organism described herein is an insect, preferably Spodoptera frugiperda (Sf); and the virus-contaminated cell population is an established cell line or cell strain, such as a commercially available or commercially available cell line, preferably an insect cell line, more preferably an Sf cell line (e.g., Sf9 cell line, Sf21 cell line, etc.).

[0037] Therefore, the cell line of the present invention may be directly or indirectly derived from Spodoptera frugiperda, or may be derived from commercially available Sf9 or Sf21 cell lines.

[0038] Furthermore, the present invention provides a virus-free Sf cell line (referred to as SF-RVN cell line in the present invention) in another aspect, which is derived from a commercially available Sf9 or Sf21 cell line and established by the above method.

[0039] Compared with the prior art, the present invention has the following beneficial technical effects:

[0040] Currently, the primary method for inhibiting Sf-Rhabdovirus in Sf9 cells is to use nucleoside chemicals. However, these drugs are broad-spectrum antiviral agents. Adding nucleoside drugs to cultured cells may not only inhibit the virus but also hinder the synthesis, transcription, and metabolism of the cell's own genes, causing some damage to the cells. The present invention, however, provides a nucleic acid molecule or combination thereof that reduces and eliminates Sf-Rhabdovirus contamination in Sf9 cells. It also uses RNA interference to specifically inhibit Sf-Rhabdovirus, minimizing damage to the cells themselves and effectively inhibiting viral replication, resulting in a stable, virus-free cell line. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Specific DNA templates obtained by PCR. PCR was performed on cDNA obtained by reverse transcription of Sf-Rhabdovirus mRNA using the eight designed specific primers. The products are shown in the figure. M represents the marker (Trans 2K DNA Marker, Quanshi Gold, BM101-01), 1 represents dsRNA-N, 2 represents dsRNA-P, 3 represents dsRNA-M, 4 represents dsRNA-G, 5 represents dsRNA-L1, 6 represents dsRNA-L2, 7 represents dsRNA-L3, and 8 represents dsRNA-L4.

[0042] Figure 2 Specific T7 DNA templates were obtained by PCR. PCR was performed on each of the eight specific DNA templates using the eight designed T7 primers. The products are shown in the figure. M represents the marker (Trans 2K DNA Marker, Quanshi Gold, BM101-01), 1 represents dsRNA-N-T7, 2 represents dsRNA-P-T7, 3 represents dsRNA-M-T7, 4 represents dsRNA-G-T7, 5 represents dsRNA-L1-T7, 6 represents dsRNA-L2-T7, 7 represents dsRNA-L3-T7, and 8 represents dsRNA-L4-T7.

[0043] Figure 3 dsRNA is obtained by in vitro transcription using a specific T7 DNA template. Single-stranded RNA is transcribed in vitro using DNA as a template. The two complementary single-stranded RNAs are then combined into a single long dsRNA through heating (denaturation) and annealing. Purification then yields dsRNA suitable for cell transfection. (A) dsRNA-L3-T7 DNA template is transcribed in vitro to obtain the corresponding dsRNA. M represents a marker (Trans 2K DNA Marker, gold, BM101-01), 1 represents L3-dsRNA (before denaturation), 2 represents L3-dsRNA (after annealing), and 3 represents L3-dsRNA (after purification). (B) dsRNA is obtained by in vitro transcription using a T7 DNA template. M represents a marker (Trans 2K DNA Marker, gold, BM101-01), 1 represents L1-dsRNA (after purification), 2 represents L2-dsRNA (after purification), and 3 represents L4-dsRNA (after purification). (C) T7 DNA template was used for in vitro transcription to obtain the corresponding dsRNA. M represents marker (Trans 2K DNA Marker, gold, BM101-01), 1 represents N-dsRNA (purified), 2 represents P-dsRNA (purified), 3 represents M-dsRNA (purified), and 4 represents G-dsRNA (purified). DETAILED DESCRIPTION

[0044] To facilitate understanding of various embodiments of the present disclosure, the following explanations of specific terms are provided.

[0045] Cell line: refers to a cell population expanded from one or more common ancestral cells, including but not limited to a cell population expanded from a single isolated cell.

[0046] Established cell lines: These are cell lines that have the potential to proliferate indefinitely when cultured under appropriate conditions. These cell lines have undergone changes (e.g., transformation) in vitro compared to naturally occurring cells in an organism. A second cell line obtained by isolating single cells from a first cell line and then expanding the isolated cells is sometimes referred to as a subclone of the first cell line.

[0047] Derived from an organism: refers to cells obtained directly or indirectly from an organism. Cells can be directly derived from an organism, for example, by obtaining a tissue or organ from the organism and then lysing the tissue or organ to obtain primary cells. Cells can also be indirectly derived from an organism, for example, by isolating a single cell from a cell line derived from the organism and then amplifying the isolated single cell to establish and obtain a cell line.

[0048] The organisms described herein can be insects, particularly Lepidoptera. Lepidoptera refers to any member of the order Lepidoptera, including butterflies and moths, whose adults have four broad or lanceolate wings, typically covered with slightly overlapping, brightly colored scales, and whose larvae are caterpillars. For example, Lepidoptera include, but are not limited to, Spodoptera frugiperda, Trichoplusia ni, or Bombyx mori.

[0049] Single-cell clones are cell populations formed by the culture and expansion of a single cell. Multi-cell clones are cell populations formed by the culture and expansion of multiple cells.

[0050] dsRNA (double-stranded RNA) or double-stranded RNA refers to a double-stranded RNA molecule composed of two reverse complementary RNA sequences. After entering the cell, it can be cut into siRNA, thereby inducing RNA interference.

[0051] siRNA (small interfering RNA), also known as small interfering RNA or short interfering RNA, is a type of double-stranded RNA with a length of generally 19-25 base pairs. It mainly participates in RNA interference, causing mRNA level degradation and leading to the obstruction of protein translation.

[0052] "Comprising" or "including" are synonymous and are open-ended terms that do not exclude the presence of other unrecited components, elements, steps, etc. For example, a composition "comprising" components A, B, and C can consist of components A, B, and C; or the composition can contain not only components A, B, and C, but also one or more other components.

[0053] The terms "detecting the presence of a virus," "detecting the presence of an Sf-Rhabdovirus," and related terms are used extensively throughout this specification. Those skilled in the art will appreciate that many detection techniques are known in the art and can be used in the present invention. Exemplary techniques for detecting viruses include polymerase chain reaction (PCR), reverse transcription (RT), reverse transcription-polymerase chain reaction (RT-PCR), RT-PCR combined with nested PCR, quantitative PCR (Q-PCR), RT-PCR combined with quantitative PCR (quantitative RT-PCR or RT-QPCR), various probe hybridization techniques, electron microscopy, and various antibody-based detection techniques known in the art (e.g., ELISA assays). Detection techniques also include, but are not limited to, plaque assays and cytopathic effect (CPE) observations, as well as bioinformatics techniques such as BLAST searches.

[0054] The present invention provides a nucleic acid molecule or a combination thereof that can inhibit Sf-Rhabdovirus, or can reduce and eliminate Sf-Rhabdovirus contamination in cell lines, including deoxyribonucleic acid (RNA) or a combination thereof and ribonucleic acid (DNA) or a combination thereof.

[0055] In one embodiment of the present invention, RNA includes dsRNA and siRNA.

[0056] In another embodiment of the present invention, the sequence of a dsRNA is any one of SEQ ID NOs: 1-8 or a combination thereof. In a specific embodiment of the present invention, the dsRNA sequence refers to a sequence in a dsRNA molecule that is formed by combining (e.g., covalently linking) two or more of the sequences shown in any one of SEQ ID NOs: 1-8.

[0057] In a specific embodiment of the present invention, the sequence of the dsRNA is preferably the sequence shown in SEQ ID NO: 3.

[0058] In one embodiment of the present invention, the above-mentioned dsRNA sequence is: a sequence composed of SEQ ID NO: 1 and SEQ ID NO: 3; a sequence composed of SEQ ID NO: 2 and SEQ ID NO: 3; a sequence composed of SEQ ID NO: 3 and SEQ ID NO: 4; a sequence composed of SEQ ID NO: 3 and SEQ ID NO: 8; a sequence composed of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 8; or a sequence composed of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0059] In a specific embodiment of the present invention, the dsRNA sequence is preferably a combination of the sequence shown in SEQ ID NO: 3 and other sequences.

[0060] In certain embodiments, the dsRNA is a modified dsRNA obtained by modifying the nucleotides of the above sequence. Such modifications include, but are not limited to, chemical modifications to improve the in vivo stability of the dsRNA. Specific types and methods of modification are well known to those skilled in the art.

[0061] The present invention also relates to a dsRNA combination, i.e., a combination of any two or more dsRNAs disclosed herein. In one embodiment of the present invention, the dsRNA combination refers to any two or more dsRNAs used or applied in conjunction or combination in the same use, method, or kit.

[0062] In a specific embodiment of the present invention, the dsRNA combination is: a combination of dsRNAs set forth in SEQ ID NO: 1 and SEQ ID NO: 3; a combination of dsRNAs set forth in SEQ ID NO: 2 and SEQ ID NO: 3; a combination of dsRNAs set forth in SEQ ID NO: 3 and SEQ ID NO: 4; a combination of dsRNAs set forth in SEQ ID NO: 3 and SEQ ID NO: 8; a combination of dsRNAs set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 8; or a combination of dsRNAs set forth in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0063] In a specific embodiment of the present invention, a dsRNA combination is preferably a combination of a dsRNA having a sequence shown in SEQ ID NO: 3 and other dsRNAs of the present invention.

[0064] The present invention also provides siRNA molecules obtained by cleaving the above-mentioned dsRNA or dsRNA combination.

[0065] In another embodiment of the present invention, the siRNA may consist of any 14-30 consecutive nucleotides within any one of the sequences shown in SEQ ID NOs: 1-8 or a combination thereof, preferably 16-25 consecutive nucleotides, more preferably 18-21 consecutive nucleotides.

[0066] The present invention also provides a DNA encoding the above dsRNA.

[0067] In certain embodiments of the present invention, the DNA sequence encoding the dsRNA is any one of SEQ ID NOs: 9-16 or a combination thereof. In one embodiment of the present invention, the DNA sequence encoding the dsRNA is a sequence in a DNA molecule formed by combining (e.g., covalently linking) two or more of the sequences shown in any one of SEQ ID NOs: 9-16.

[0068] In a specific embodiment of the present invention, a DNA sequence encoding the above dsRNA is preferably the sequence shown in SEQ ID NO: 11.

[0069] In one embodiment of the present invention, a DNA sequence encoding the above-mentioned dsRNA is: a sequence combined with SEQ ID NO: 9 and SEQ ID NO: 11; a sequence combined with SEQ ID NO: 10 and SEQ ID NO: 11; a sequence combined with SEQ ID NO: 11 and SEQ ID NO: 12; a sequence combined with SEQ ID NO: 11 and SEQ ID NO: 16; a sequence combined with SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 16; or a sequence combined with SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0070] In a specific embodiment of the present invention, the DNA sequence encoding the above dsRNA is preferably a combination of the sequence shown in SEQ ID NO: 11 and other sequences.

[0071] In certain embodiments, the DNA is modified DNA obtained by modifying the nucleotides of the above sequence. The modification includes but is not limited to chemical modification that can improve the stability of DNA in vivo. The specific modification types and modification methods are well known in the art.

[0072] The present invention also relates to a combination of DNAs, i.e., a combination of any two or more DNAs disclosed herein. In one embodiment of the present invention, the combination of DNAs refers to any two or more DNAs used or applied in conjunction or combination in the same application, method, or kit.

[0073] In certain embodiments of the present invention, a combination of the above-mentioned DNAs is provided, wherein the combination is: a combination of DNAs set forth in SEQ ID NO: 9 and SEQ ID NO: 11; a combination of DNAs set forth in SEQ ID NO: 10 and SEQ ID NO: 11; a combination of DNAs set forth in SEQ ID NO: 11 and SEQ ID NO: 12; a combination of DNAs set forth in SEQ ID NO: 11 and SEQ ID NO: 16; a combination of DNAs set forth in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 16; or a combination of DNAs set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0074] In a specific embodiment of the present invention, the DNA combination is preferably a combination of the DNA of the sequence shown in SEQ ID NO: 11 and other DNAs of the present invention.

[0075] In certain embodiments of the present invention, the DNA is a DNA encoding the above-mentioned siRNA.

[0076] The present invention also provides a delivery vector comprising the above-mentioned nucleic acid molecules (including RNA or DNA) or a combination thereof, which is used to induce gene silencing or RNA interference. The delivery vector is selected from a viral vector and a non-viral vector. Preferably, the non-viral vector is selected from the group consisting of liposomes, plasmid vectors, and phage vectors. Preferably, the viral vector is selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, and hybrid viral vectors. More preferably, the viral vector is selected from an adeno-associated viral vector. In one embodiment of the present invention, the delivery vector comprises the above-mentioned dsRNA or dsRNA combination or DNA or DNA combination. In another embodiment of the present invention, different dsRNA or DNA molecules in the dsRNA or DNA combination are in the same delivery vector and in the same expression frame. In another embodiment of the present invention, different dsRNA or DNA molecules in the dsRNA combination or DNA combination can be in the same expression frame or in different expression frames. In another embodiment of the present invention, different dsRNA or DNA molecules in the dsRNA combination or DNA combination can be in the same delivery vector or in different delivery vectors.

[0077] In another aspect, the present invention provides a kit for inducing RNA interference. In a specific embodiment of the present invention, the kit comprises the above-mentioned dsRNA or a combination thereof, DNA or a combination thereof, siRNA and / or a delivery vector.

[0078] In another aspect, the present invention provides a method for establishing and obtaining an Sf-Rhabdovirus-free cell line, utilizing the aforementioned nucleic acid molecules (including RNA or DNA) or a combination thereof, or a delivery vector comprising the aforementioned nucleic acid molecules (including RNA or DNA) or a combination thereof, to reduce and ultimately eliminate Sf-Rhabdovirus contamination in cells through RNA interference technology.

[0079] Certain exemplary methods for obtaining virus-free cell lines include: obtaining cells from a virus-contaminated organism or cell population; washing the cells, and then introducing the dsRNA or a combination thereof, or DNA or a combination thereof, or a delivery vector comprising the RNA / RNA combination or DNA / DNA combination of the present invention into the obtained cells to induce RNA interference; washing and resuspending the introduced cells and then separating them into single cells or multiple cells; culturing and expanding the separated single cells or multiple cells in a culture medium suitable for cell growth and division to form single-cell clones or multi-cell clones, thereby obtaining a virus-free cell line.

[0080] In certain embodiments, the method for obtaining a virus-deficient cell line further comprises removing a portion from the cell clone or the culture medium to detect the presence of the virus.

[0081] In certain embodiments, the virus contaminating an organism or cell population described herein is an Sf-rhabdovirus. In some embodiments, the virus-contaminated organism described herein is an insect, preferably Spodoptera frugiperda; and the virus-contaminated cell population described herein is an established cell line or cell strain, such as a commercially available or commercially available cell line, preferably an insect cell line, more preferably an Sf9 or Sf21 cell line.

[0082] In certain embodiments, the technology of RNA interference using nucleic acid molecules is well known in the art. The methods of delivering nucleic acid molecules in RNA interference mainly include delivery through non-viral vectors and / or through viral vectors, wherein non-viral vectors include but are not limited to liposomes, plasmid vectors, phage vectors or bacterial vectors; viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, chronic disease vectors or hybrid viral vectors.

[0083] In certain embodiments, a single cell refers to only one cell; a plurality of cells refers to a plurality of cells, including but not limited to, 2-20 cells, preferably 3-10 cells, preferably 4-6 cells, and more preferably 5 cells.

[0084] In certain embodiments, methods of separation into single cells or multiple cells include, but are not limited to, limiting dilution cloning (serial dilution cloning), cloning cells in soft agar and then picking cell colonies, cell sorting, laser capture microdissection (LCM), manual capture using a micropipette, microfluidics, or use of a micromanipulator.

[0085] In certain embodiments, methods for detecting the presence of viruses (including but not limited to Sf-Rhabdovirus) are well known in the art, including but not limited to RT-PCR, nested PCR, RT-PCR combined with nested PCR, or RT-QPCR.

[0086] In certain embodiments, after the cell clones detected for the absence of the virus are expanded and cultured in normal culture medium for a period of time, part of the expanded culture cells and the supernatant culture fluid are taken to verify again whether the virus has been completely eliminated.

[0087] In certain embodiments, the virus-free cell lines established are derived from primary cells contaminated by viruses (including but not limited to Sf-Rhabdovirus). In certain embodiments, the virus-contaminated primary cells are derived from virus-contaminated organisms (including but not limited to Spodoptera frugiperda).

[0088] In other embodiments, the virus-free cell line is derived from a cell population (including but not limited to commercially available or commercially available cell lines) contaminated with a virus (including but not limited to Sf-Rhabdovirus). In certain embodiments, the virus-contaminated cell population is an Sf cell line contaminated with Sf-Rhabdovirus, including but not limited to Sf9 or Sf21 cell lines.

[0089] It will be appreciated by those skilled in the art that materials and conditions suitable for the growth and incubation of specific cell types are known in the art, and sources for obtaining such materials and conditions are also known to those skilled in the art. Such sources include, but are not limited to, cell culture manuals, commercial cell banks, or culture medium suppliers. Suitable cell culture conditions can also be readily determined using methods known in the art.

[0090] The present invention will be further described below by way of specific examples. It should be understood that the following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0091] The techniques used in the following examples, including biological techniques such as gene sequencing, PCR amplification and detection, cell transfection, and cell culture, are conventional techniques known to those skilled in the art unless otherwise specified. The instruments, equipment, and reagents used are all publicly available to those skilled in the art, unless otherwise specified in this specification.

[0092] Example 1 DNA template preparation

[0093] Based on the conserved sequences in the Sf-Rhabdovirus genome (GenBank accession number KF947078.1), eight long dsRNA sequences of 500-900 bp were designed and obtained that specifically targeted the Sf-Rhabdovirus genome and did not target the Sf21 or Sf9 cell genomes. The specific sequences are shown in Table 1, and the corresponding DNA sequences are shown in Table 2.

[0094] Table 1 dsRNA sequences

[0095]

[0096]

[0097]

[0098] Table 2 dsRNA corresponding to DNA

[0099]

[0100]

[0101]

[0102] The total mRNA of Sf9 cells and Sf-rhabdovirus was extracted using RNeasy Plus Mini kit (Qiagen, 74134), and then the mRNA was reverse transcribed to synthesize cDNA (the kit used was III First-Strand Synthesis System for RT-PCR, Invitrogen, 18080051).

[0103] The cDNA was amplified by PCR using 8 pairs of designed and synthesized specific primers (specific sequences are shown in Table 3) (the kit used was Pyrobest DNA polymerase, Takara, R005A), and 8 specific DNA templates were amplified (the reaction program was 94°C for 3 min; 28 cycles for 94°C for 30 s, 60°C for 30 s, 72°C for 1 min; 72°C for 10 min; 8°C + ∞). The PCR amplification results are shown in Figure 1 , and recover the DNA templates respectively.

[0104] Table 3 dsRNA primer design

[0105]

[0106]

[0107] Eight specific DNA templates (dsRNA-N, dsRNA-P, dsRNA-M, dsRNA-G, dsRNA-L1, dsRNA-L2, dsRNA-L3, and dsRNA-L4) were ligated to T-vectors and sequenced. Sequencing results showed that the synthesized specific DNA templates matched sequences found in the Sf-Rhabdovirus genome.

[0108] The 8 pairs of specific T7 primers designed and synthesized (specific sequences are shown in Table 4) were used to perform PCR amplification on the products recovered from the specific DNA templates (the kit used was Pyrobest DNA polymerase, Takara, R005A), and 8 specific T7 DNA templates were amplified (the reaction program was 94°C for 3 min; 30 cycles for 94°C for 30 s, 58°C for 30 s, 72°C for 1 min; 72°C for 10 min; 8°C + ∞). The PCR amplification results are shown in Figure 2 , the product was recovered and used for dsRNA synthesis.

[0109] Table 4 dsRNA T7 primer design

[0110]

[0111]

[0112] Example 2 In vitro transcription synthesis of dsRNA

[0113] In vitro synthesis of dsRNA: including in vitro transcription, denaturation annealing, removal of impurities such as DNA and ssRNA, purification, nucleic acid gel electrophoresis, etc., see MEGAscript for details TM RNAi kit instructions (Invitrogen, AM1626), and finally obtained 8 high-concentration, relatively single-band dsRNAs that can be used for transfection (Table 5, Figure 3 ).

[0114] Table 5 Concentrations of the 8 synthesized dsRNAs

[0115]

[0116] Example 3 Sf-Rhabdovirus Detection Method

[0117] To verify the presence of Sf-Rhabdovirus in cells and culture supernatant, RNA was extracted from the supernatant of transfected cells and cells and supernatants at different passages, and RT-PCR was used to detect Sf-Rhabdovirus (see: Ma et al., J. Virol. 88:6576-85, 2014). Furthermore, to more accurately and precisely detect the presence of Sf-Rhabdovirus, a set of primers and probes were designed based on the Sf-Rhabdovirus L sequence (Genbank: KF947078.1) using Primer Premier 6.25. One-step quantitative RT-PCR (RT-QPCR) was used to quantify the Sf-Rhabdovirus content. RNA was first extracted from cells and culture supernatant, and then one-step RT-QPCR was performed using the TaqMan RNA-to-CT 1-Step Kit (ABI / Thermo, 4392938) according to the manufacturer's instructions to detect Sf-Rhabdovirus content.

[0118] Example 4 dsRNA transfection of cells

[0119] Sf9 cells were cultured in serum-free medium (ESF AF, Expression Systems). First, eight synthesized dsRNAs and transfection reagents (Cellfectin TM II Reagent, Invitrogen, 10362100) was used for single transfection. The transfection conditions refer to Cellfectin TMII Reagent Kit (Invitrogen, 10362100) was used as described in the instructions. Two days after cell transfection, the Sf-Rhabdovirus content in the culture supernatant was measured using the same assay as described in Example 3. RT-PCR assays revealed that each of the eight dsRNAs significantly reduced Sf-Rhabdovirus content compared to untransfected Sf9 cells (results not shown). RT-QPCR results are shown in Table 6, where + indicates a positive control without dsRNA.

[0120] Table 6 Sf-Rhabdovirus content in the supernatant of cells transfected with 8 dsRNAs alone

[0121]

[0122] The results showed that the eight designed and synthesized dsRNAs had a significant inhibitory effect on Sf-Rhabdovirus after being transfected into Sf9 cells alone. The content of Sf-Rhabdovirus in the cell supernatant was significantly reduced, among which M-dsRNA had the most obvious inhibitory effect, decreasing by 67.8%.

[0123] Subsequently, transfection was performed using multiple dsRNA combinations, and the Sf-Rhabdovirus content in the supernatant culture medium was assayed to verify the inhibitory effect of the dsRNA combination on Sf-Rhabdovirus, using the same assay method as described in Example 3. RT-QPCR test results are shown in Table 7, where + indicates a positive control without dsRNA addition.

[0124] Table 7 Sf-Rhabdovirus content in the supernatant of cells transfected with different dsRNA combinations

[0125]

[0126] The results showed that compared to single dsRNAs, different dsRNA combinations further improved the inhibitory effect against Sf-Rhabdovirus, with M+P reducing activity by 76% and M+N+P+G+L4 reducing activity by 75%. Ultimately, M+P and M+N+P+G+L4 were selected for further research and validation.

[0127] Example 5 Screening of Sf-virus-free cell clones and identification of Sf-rhabdovirus

[0128] Sf9 cells were transfected with either M+P or M+N+P+G+L4, respectively. The cells were washed and then transfected. Two days after transfection, the cells were washed, resuspended, and isolated by limiting dilution into single cells (1 cell / well) or multiple cells (5 cells / well). The cells were expanded and cultured in normal culture medium to form single-cell and multi-cell clones. Samples were then collected for Sf-Rhabdovirus testing, as described in Example 3.

[0129] Finally, the M+P combination screened out 1 Sf-Rhabdovirus-free cell line (from a single cell clone of 1 cell / well), while the M+N+P+G+L4 combination screened out 10 Sf-Rhabdovirus-free cell lines (3 of which were from a single cell clone of 1 cell / well and 7 were from multi-cell clones of 5 cells / well).

[0130] Two of the selected Sf-Rhabdovirus-free cell lines, 38C3 and 38B4 (both from polyclonal clones with 5 cells / well), were selected for further validation. The 38C3 and 38B4 cells were further expanded in culture. The P0 generation was defined as the time when the cells were expanded into T25 flasks. The cells and supernatant culture medium of different generations were assayed for Sf-Rhabdovirus using the same assay method as described in Example 3. The results of the Sf-Rhabdovirus assay are shown in Table 8.

[0131] Table 8 Screened Cells Sf-Rhabdovirus Detection

[0132]

[0133] In summary, the designed long-chain dsRNA effectively inhibited Sf-Rhabdovirus and established a stable Sf-Rhabdovirus-free cell line (SF-RVN cells). This conclusion was based on highly sensitive RT-PCR and RT-QPCR results. These methods demonstrated that Sf-Rhabdovirus was not detected in SF-RVN cells (a screened Sf-Rhabdovirus-free cell line) or in the supernatant culture medium after at least 17 passages.

[0134] Functional studies of the screened SF-RVN cells revealed that SF-RVN cells and Sf9 cells shared similar basic cell growth characteristics, including cell viability, cell diameter, and cell aggregation rate (results not shown). SF-RVN cells and Sf9 cells were essentially equivalent in terms of baculovirus passage stability and recombinant AAV virus production (results not shown), indicating that the methods of the present invention did not affect the activity of Sf9 cells.

[0135] The above results show that the screened SF-RVN cells can completely replace Sf9 cells and become safe production cells for proteins, viruses and vaccines. Sequence Listing <110> Shutaishen (Beijing) Biopharmaceutical Co., Ltd.; Beijing Sinocare Biotechnology Co., Ltd. <120> A nucleotide sequence for inducing RNA interference and reducing and eliminating viral contamination in cells and its application <160> 48 <210> 1 <211> 524 <212> RNA <213> Artificial Sequence <400> 1 ggaguguuga uacaugucgg gagaucaguu uaaaucugaa guuaccuggu gaaauauggc 60 aacuggccca ucaagaaacc aucuucaaca gauuucuuac auuuuacgcu acuggguaug 120 uuccaaauac acacacagcc acagaaauug uacucuccau ggcaucacua aucuucaagg 180 acaaggccaa agcaccuauu gauuugauuu gggaugacuc auuucaagcu agucccucug 240 aggagugugg guucuccguu guuggagaaa cuccauuggu uaucggacaa cacccggaug 300 augaugacua cacauugaga gaagaugaag aaucagccgc uaugaaugag gaagaaaaaa 360 uacaagcagc ucuaaaaacu uugggaauuc aagauacucc aguagaccug aaggaugcau 420 cuggaauugu cuuugagaca aaggaggaca gagaacaaag gaucaagaau gagaaagcuc 480 uacauguaga ggaugauauc aacgcucuaa cucagauuac aaaa 524 <210> 2 <211> 588 <212> RNA <213> Artificial Sequence <400> 2 uccucucaag agaaacuauc aacugauuua cucuauaaug gcuucccacu cucuugacac 60 cauugaucua ucugaaauug gauugacaag ggagguucug acugggguug gcgauuacau 120 gacuggacaa agaccgguuc cagccuucaa uccuccagag gucggucacu cccccucuga 180 ugaaguggca aaacgauugg gagaacugaa gaauuacugg acucaguuag aggauccucu 240 ugaugagaga auucucaaua ccuugaaagc gaucagcauc cugagcggag acaccagagg 300 agaucugagu ggaaaauaua aacaucuagu ccgcauuagc ggagaugaca ugccccaauu 360<​​​​​​​​​​​​​​​​​​gacaucaauc auaacuuucc ucggauucuc agauggaucc ugcaucaacu cagaagcaga 60 gccaagacag cucacaggau cuagauccug ggagaucaug ucuccuaauc agaaucucau 120 ugugauaacc cuaggauuca aaauaaccuu gaaaaccuuc gcacagcacc agagauacag 180 cuugcgugac cauggauucc acaaauugga gaugcucaac gagaaagaga agaaaauguu 240 gaacuauaug ggggucaaac aauuaaaacc ccaguauaca caugaaaaga cauucgagaa 300 acucauucuc aagaacaaag guccaaaggg gucucguguc agggcaauuc uucacucuca 360 aagucgugac auguggucuc caaccgcucc uucuccucca cccacauaug aagauggauc 420 cucagaugaa ugggaucagc aacaacugca cagccucaac caccugcaua caccuucugu 480 cccccugagg gcccccagga cauccccacc ccaacaacuc uccccaaaac cgacauccac 540 aacccaaccc cucccacaac ucacacaacc aaacaagccc caagaac 587 <210> 4 <211> 547 <212> RNA <213> Artificial Sequence <400> 4 uuauggcaag agagaauuga caccacuugc uccuggaacu ucuggggcaa uuacaaagga 60 uccauuguau cuaaauccuc aguaccucua aaggauaucc caucggguag ugcccggaau 120 ggauauuggg cuuugagcaa ugaugaaguu caagagauug aucauguccc uuacaacuug 180 agauauuauu guuacuggug cagaaaugaa uauccuggga gcuuuuauau gagauaugua 240 aagaaaguuc ggaucauaag aaauccugau gggucuauaa agacuccuag aggauccugg 300 guucaugagu uggacaacuu guggggagau cagaugaggu aucuaguuau ucgaagauuu 360 gggggagaau cuagcugccc ucuuaagaua uaugauguga gagcaggggu ucugucaaaa 420 ucucggucaa acuucaucuu agugucccuu cccuccuuga auuugcaguu cucuguauca 480 cuugaaucca cugagacgaa augcucauuu ggagauaaga cauaugauau ugugcagagc 540 augggag 547 <210> 5 <211> 682 <212> RNA <213> Artificial Sequence <400> 5 caauucauac aucccaauaa ccccauucga cgagaguacu uggagaugca gagacaacuu 60 cagauaacac cccccaaucu auuugaucua ucaaaaguuc aggguuuuuu ccuaaaugug 120 uuuaauguac cagucucuag ccuuccuuua uuagaauuua gacaagcauu gcacuuggcu 180 ucucaacuau accaaguaga aguugaaggg guucucaaag agcuaggggc aucagcuacu 240 aaaauugaua uaucuccucu gaugaaaaau aaggacuuaa uuaaucuuua ucugagaaaa 300 uguuucuggg aggaagcagu ugucaugagu ggaaaugaua acucuaguca gggauccugg 360 uggucaagag cagauaaagg gcuuauucuc uuuagacgac cugggcuuga uaucauaauu 420 ggggagaauu uaaugucaau ccagacaucu cagaacucca uauuggucuc ccgagaucac 480 cuaaccauau ugucagaucu cgcugcugag cgguuuagua uaauucucca auccuucuua 540 gcugaucaaa cccauaauac agauaugccc accccuuccg aauuaaguuu auuucuuaag 600 gaaggagaug aaaugcuaac uuuagcagga aaucaaggau augaucuaau uuauacuuua 660 gaaucuuccu guacuucccg au 682 <210> 6 <211> 855 <212> RNA <213> Artificial Sequence <400> 6 uaugaaggag gcucguggaa agauucuaaa uuccggcaug aaauuguuaa agauuuagag 60 120 ucaguuuuuug aacgaaaucu aaacgccuuc acccaacugu augggcuaua ucgcauaugg 180 ggucacccaa cucuggaucc auuacuuggg acaauagccc ucaaagaauu gggaacaaca 240 ccaagauugu accuaucaca ccaagcucag gagauuaaca acaaguuuaa ggagaaguuc 300 auaaaaagau auuaaauag acauaaggag uggccggaau uagauguauc gaaauuacca 360 agacauaaca ucauucgagu ccauuaugag aagaaauuac aauuuuccuuc uaaauccaga 420 caauaagga gaucucaucu cuccuuggua gaauucaaag agguauuccc uguugauccu 480 540 cuuaacgaga ucuauagaaa caagaguauc gggaauucac uagcaagauc cuuauugcuu 600 aauuuccucu ccucugacau uucagacccc caagaauuuc ugaagaauau agauaccuca 660 ggguuuccuc cugaagagau uuguuguugg guaacgaaa aagagagaga aggaaagcua 720 aaggcaaggc uguuuggauu acugaccuua gugaaacgau cauauguagu uaucacagaa 780 aaacucuugg cugagcaucu auuuccguau uucccugaaa uaaccaugac ggaugacgag 840 uuaguuuugg agaaa 855 <210> 7 <211> 705 <212> RNA <213> Artificial Sequence <400> 7 ccaaguggaa caccaauaug agagccccag acacacagcc auuuuaccac acuauagaua 60 cgauguuugg uuuggaaaau uguuuuacca ggacacauga aauguucuac aauuccuuuu 120 uguaccuuau agacgguucu uaucucccaa caauaguuga ugauggguuc aaaacagaua 180 uuggauguug gcgacaucau cuugggggaa ucgaaggucu cagacaaaaa ggauggacuc 240 uguggacagu uauguugauc aggcuaguug cggaaaaaua uauuuucaau augucuauca 300 ugggacaggg ggacaaucaa augcuacuuc uaacuuucga uucuaauacc ccggaagaau 360 augcccucuc ucaaguuaau gauuuccuuc agucauuaaa ggauaaacug ucacuaauag 420 guccuccucu caaguuggag gaaacuugga uuuccaaaga cuuuuauuua uauggaaagu 480 auccuaucaa aggagguguu ucucucacca caucguggaa aaaaucaugc agaauguucc 540 gauguugcaa cgaggacuau cccaccauag aguccaguuu guccuccuua gcugcaaacc 600 uguacucugc aguggcugcu gauaacuuua cacagacucu guuuuuuguu uacuuauuug 660 aauuaguagg ucuauuccaa ugcaauauua gaagacccua ucucc 705 <210> 8 <211> 602 <212> RNA <213> Artificial Sequence <400> 8 auuccaaugc aauauuagaa gacccuaucu ccaaaagaac ucauuuuauc aaucguuaga 60 ucgaaauaga accuucacag uugcuucugc aaaagaccaa aagaagaaac uucauguccc 120 ucuuguucua ucacccccaa aucagcuaca gccuaccgag guuuuguuag gacuauguuu 180 gacuccgagg acuuugggag gauauccagu uguucuguac ccaucggucu ugauaaaggg 240 agccccagac caauuaucau uugaucuugc guccuuaaaa uuauuuucaa agucagcaga 300 ugcaacuguu aauaggauaa uaacccgugu auccagucca uuccucuccg aguauaagaa 360 uuauucucua cuuuuuauga acccugaggc aauuauccug gagucuacac ccacuccugc 420 agaggcaagg agaacuacga uguuagaauu ucuuuccaac agugaucgug uuaaccagcc 480 uuacauaaaa gaauuccuaa acaucauuca ugagaaugca aaucaaucua uggaagauuu 540 uuuaaccuca aauccuguac uucauccacg uguaaucucu cuucuacuuc aggcaacucc 600 ac 602 <210> 9 <211> 524 <212> DNA <213> Artificial Sequence <400> 9 ggagtgttga tacatgtcgg gagatcagtt taaatctgaa gttacctggt gaaatatggc 60 aactggccca tcaagaaacc atcttcaaca gatttcttac attttacgct actgggtatg 120<00,00439>ttccaaatac acacacagcc acagaaattg tactctccat ggcatcacta atcttcaagg 180 acaaggccaa agcacctatt gatttgattt gggatgactc atttcaagct agtccctctg 240 aggagtgtgg gttctccgtt gttggagaaa ctccattggt tatcggacaa cacccggatg 300 atgatgacta cacattgaga gaagatgaag aatcagccgc tatgaatgag gaagaaaaaa 360 tacaagcagc tctaaaaact ttgggaattc aagatactcc agtagacctg aaggatgcat 420 ctggaattgt ctttgagaca aaggaggaca gagaacaaag gatcaagaat gagaaagctc 4,80 tacatgtaga ggatgatatc aacgctctaa ctcagattac aaaa 524 <210> 10 <211> 588 <212> DNA <213> Artificial Sequence <400> 10 tcctctcaag agaaactatc aactgattta cctataatg gcttcccact ctcttgacac 60 cattgatcta tctgaaattg gattgacaag ggaggttctg actggggttg gcgattacat 120 gactggacaa agaccggttc cagccttcaa tcctccagag gtcggtcact ccccctctga 180 tgaagtggca aaacgattgg gagaactgaa gaattactgg actcagttag aggatcctct 240 tgatgagaga attctcaata ccttgaaagc gatcagcatc ctgagcggag acaccagagg 300 agatctgagt ggaaatata aacatctagt ccgcattagc ggagatgaca tgccccaatt 360 attggacgaa cttatagaca tctgtcttct ggggcctaag actctaattg ctaccttacg 420 aatggcgata accgcctata ccgctgcatt agccagaaat gccaagtcca ccatctcaga 480 tattactacc gcatcagcag atttgatggt catcactcag atgatacagt cccagcagga 540 atctttccaa tcatcattag agcatctctc tcatgcttgg aataacgt 588 <210> 11 <211> 587 <212> DNA <213> Artificial Sequence <400> 11 gacatcaatc ataactttcc tcggattctc agatggatcc tgcatcaact cagaagcaga 60 gccaagacag ctcacaggat ctagatcctg ggagatcatg tctcctaatc agaatctcat 120 tgtgataacc ctaggattca aaataacctt gaaaaccttc gcacagcacc agagatacag 180 cttgcgtgac catggattcc acaaattgga gatgctcaac gagaaagaga agaaaatgtt 240 gaactatatg ggggtcaaac aattaaaacc ccagtataca catgaaaaga cattcgagaa 300 actcattctc aagaacaaag gtccaaaggg gtctcgtgtc agggcaattc ttcactctca 360 aagtcgtgac atgtggtctc caaccgctcc ttctcctcca cccacatatg aagatggatc 420 ctcagatgaa tgggatcagc aaactgca cagcctcaac cacctgcata caccttctgt 480 ccccctgagg gcccccagga catccccacc ccaacaactc tccccaaaac cgacatccac 540 aacccaaccc ctcccacaac tcacacaacc aaacaagccc caagaac 587 <210> 12 <211> 547 <212> DNA <213> Artificial Sequence <400> 12 ttatggcaag agagaattga caccacttgc tcctggaact tctggggcaa ttacaaagga 60 tccattgtat ctaaatcctc agtacctcta aaggatatcc catcgggtag tgcccggaat 120 ggatattggg ctttgagcaa tgatgaagtt caagagattg atcatgtccc ttacaacttg 180 agatattatt gttactggtg cagaaatgaa tatcctggga gcttttatat gagatatgta 240 aagaaagttc ggatcataag aaatcctgat gggtctataa agactcctag aggatcctgg 300 gttcatgagt tggacaactt gtggggagat cagatgaggt atctagttat tcgaagattt 360 gggggagaat ctagctgccc tcttaagata tatgatgtga gagcaggggt tctgtcaaaa 420 tctcggtcaa acttcatctt agtgtccctt ccctccttga atttgcagtt ctctgtatca 480 cttgaatcca ctgagacgaa atgctcattt ggagataaga catatgatat tgtgcagagc 540 atgggag 547 <210> 13 <211> 682 <212> DNA <213> Artificial Sequence <400> 13 caattcatac atcccaataa ccccattcga cgagagtact tggagatgca gagacaactt 60 cagataacac cccccaatct atttgatcta tcaaaagttc agggtttttt cctaaatgtg 120 tttaatgtac cagtctctag ccttccttta ttagaattta gacaagcatt gcacttggct 180 tctcaactat accaagtaga agttgaaggg gttctcaaag agctaggggc atcagctact 240 aaaattgata tatctcctct gatgaaaaat aaggacttaa ttaatcttta tctgagaaaa 300 tgtttctggg aggaagcagt tgtcatgagt ggaaatgata actctagtca gggatcctgg 360 tggtcaagag cagataaagg gcttattctc tttagacgac ctgggcttga tatcataatt 420 ggggagaatt taatgtcaat ccagacatct cagaactcca tattggtctc ccgagatcac 480 ctaaccatat tgtcagatct cgctgctgag cggtttagta taattctcca atccttctta 540 gctgatcaaa cccataatac agatatgccc accccttccg aattaagtttt atttcttaag 600 gaaggagatg aaatgctaac tttagcagga aatcaaggat atgatctaat ttatacttta 660 gaatcttcct gtacttcccg at 682 <210> 14 <211> 855 <212> DNA <213> Artificial Sequence <400> 14 tatgaaggag gctcgtggaa agattctaaa ttccggcatg aaattgttaa agatttagag 60 aaaaaagcct cagatctaaa tttacatcct caacttagag ttagagaaca actgttagat 120 tcagtttttg aacgaaatct aaacgccttc acccaactgt atgggctata tcgcatatgg 180 ggtcacccaa ctctggatcc attacttggg acaatagccc tcaaagaatt gggaacaaca 240 ccaagattgt acctatcaca ccaagctcag gagattaaca acaagtttaa ggaagagttc 300 ataaaaagat atttaaatag acataaggag tggccggaat tagatgtatc gaaattacca 360 agacataaca tcattcgagt ccattatgag aagaaattac aatttccttc taaatccaga 420 caatatagga gatctcatct ctccttggta gaattcaaag aggtattccc tgttgatcct 480 aaatttgatc ttattgaatt tattgatgat aaatccatct ccttaggttt cccagatctc 540 cttaacgaga tctatagaaa caagagtatc gggaattcac tagcaagatc cttattgctt 600[[ID=I24]]<O000523>aatttcctct cctctgacat ttcagacccc caagaatttc tgaagaatat agatacctca 660 gggtttcctc ctgaagagat ttgtgttggg gtacacgaaa aagagagaga aggaaagcta 720 aaggcaaggc tgtttggatt actgacctta gtgaaacgat catatgtagt tatcacagaa 780 aaactcttgg ctgagcatct atttccgtat ttccctgaaa taaccatgac ggatgacgag 840 ttagttttgg agaaa 855 <210> 15 <211> 705 <212> DNA <213> Artificial Sequence <400> 15 ccaagtggaa caccaatatg agagccccag acacacagcc atttaccac actatagata 60 cgatgtttgg tttggaaaat tgttttacca ggacacatga aatgttctac aattcctttt 120 tgtaccttat agacggttct tatctcccaa caatagttga tgatgggttc aaaacagata 180 ttggatgttg gcgacatcat cttgggggaa tcgaaggtct cagacaaaaa ggatggactc 240 tgtggacagt tatgttgatc aggctagttg cggaaaaata tattttcaat atgtctatca 300 tgggacaggg ggacaatcaa atgctacttc taactttcga ttctaatacc ccggaagaat 360 atgccctctc tcaagttaat gatttccttc agtcattaaa ggataaactg tcactaatag 420 gtcctcctct caagttggag gaaacttgga tttccaaaga cttttattta tatggaaagt 480 atcctatcaa aggaggtgtt tctctcacca catcgtggaa aaaatcatgc agaatgttcc 540 gatgttgcaa cgaggactat cccaccatag agtccagttt gtcctcctta gctgcaaacc 600 tgtactctgc agtggctgct gataacttta cacagactct gttttttgtt tacttatttg 660 aattagtagg tctattccaa tgcaatatta gaagacccta tctcc 705 <210> 16 <211> 602 <212> DNA <213> Artificial Sequence <400> 16 attccaatgc aatattagaa gaccctatct ccaaaagaac tcattttatc aatcgttaga 60 tcgaataga accttcacag ttgcttctgc aaaagaccaa aagaagaaac ttcatgtccc 120 tcttgttcta tcacccccaa atcagctaca gcctaccgag gttttgttag gactatgttt 180 gactccgagg actttgggag gatatccagt tgttctgtac ccatcggtct tgataaaggg 240 agccccagac caattatcat ttgatcttgc gtccttaaaa ttattttcaa agtcagcaga 300 tgcaactgtt aataggataa taacccgtgt atccagtcca ttcctctccg agtataagaa 360 ttattctcta ctttttatga accctgaggc aattatcctg gagtctacac ccactcctgc 420 agaggcaagg agaactacga tgttagaatt tctttccaac agtgatcgtg ttaaccagcc 480 ttacataaaa gaattcctaa acatcattca tgagaatgca aatcaatcta tggaagattt 540 tttaacctca aatcctgtac ttcatccacg tgtaatctct cttctacttc aggcaactcc 600 and 602 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <400> 17 caattcatac atcccaataa ccc 23 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <400> 18 atcgggaagt acaggaagat tctaaag 27 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <400> 19 tatgaaggag gctcgtggaa ag 22 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <400> 20 tttctccaaa actaactcgt catcc 25 <210> twenty one <211> twenty four <212> DNA <213> Artificial Sequence <400> twenty one ccaagtggaa caccaatatg agag 24 <210> twenty two <211> 25 <212> DNA <213> Artificial Sequence <400> twenty two ggagataggg tcttctaata ttgca 25 <210> twenty three <211> 25 <212> DNA <213> Artificial Sequence <400> twenty three attccaatgc aatattagaa gaccc 25 <210> twenty four <211> twenty four <212> DNA <213> Artificial Sequence <400> twenty four gtggagttgc ctgaagtaga agag 24 <210> 25 <211> twenty three <212> DNA <213> Artificial Sequence <400> 25 ggagtgttga tacatgtcgg gag 23 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <400> 26 ttttgtaatc tgagttagag cgttg 25 <210> 27 <211> 25 <212> DNA <213> Artificial Sequence <400> 27 tcctctcaag agaaactatc aactg 25 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <400> 28 acgttattcc aagcatgaga gagat 25 <210> 29 <211> twenty four <212> DNA <213> Artificial Sequence <400> 29 gacatcaatc ataactttcc tcgg 24 <210> 30 <211> twenty two <212> DNA <213> Artificial Sequence <400> 30 gttcttgggg cttgtttggt tg 22 <210> 31 <211> twenty four <212> DNA <213> Artificial Sequence <400> 31 ttatggcaag agagaattga cacc 24 <210> 32 <211> twenty two <212> DNA <213> Artificial Sequence <400> 32 ctcccatgct ctgcacaata tc 22 <210> 33 <211> 44 <212> DNA <213> Artificial Sequence <400> 33 taatacgact cactataggg acaattcata catcccaata accc 44 <210> 34 <211> 48 <212> DNA <213> Artificial Sequence <400> 34 taatacgact cactatagggg aatcgggaag tacaggaaga ttctaaag 48 <210> 35 <211> 43 <212> DNA <213> Artificial Sequence <400> 35 taatacgact cactatagggg atatgaagga ggctcgtgga aag 43 <210> 36 <211> 46 <212> DNA <213> Artificial Sequence <400> 36 taatacgact cactataggg atttctccaa aactaactcg tcatcc 46 <210> 37 <211> 45 <212> DNA <213> Artificial Sequence <400> 37 taatacgact cactataggg accaagtgga acaccaatat gagag 45 <210> 38 <211> 46 <212> DNA <213> Artificial Sequence <400> 38 taatacgact cactataggg aggagatagg gtcttctaat attgca 46 <210> 39 <211> 46 <212> DNA <213> Artificial Sequence <400> 39 taatacgact cactataggg aattccaatg caatattaga agaccc 46 <210> 40 <211> 45 <212> DNA <213> Artificial Sequence <400> 40 taatacgact cactatagggg agtggagttg cctgaagtag aagag 45 <210> 41 <211> 44 <212> DNA <213> Artificial Sequence <400> 41 taatacgact cactatagggg aggagtgttg atacatgtcg ggag 44 <210> 42 <211> 46 <212> DNA <213> Artificial Sequence <400> 42 taatacgact cactataggg attttgtaat ctgagttaga gcgttg 46 <210> 43 <211> 46 <212> DNA <213> Artificial Sequence <400> 43 taatacgact cactataggg atcctctcaa gagaaactat caactg 46 <210> 44 <211> 46 <212> DNA <213> Artificial Sequence <400> 44 taatacgact cactataggg aacgttattc caagcatgag agagat 46 <210> 45 <211> 45 <212> DNA <213> Artificial Sequence <400> 45 taatacgact cactataggg agacatcaat cataactttc ctcgg 45 <210> 46 <211> 43 <212> DNA <213> Artificial Sequence <400> 46 taatacgact cactataggg agttcttggg gcttgtttgg ttg 43 <210> 47 <211> 45 <212> DNA <213> Artificial Sequence <400> 47 taatacgact cactatagggg attatggcaa gagagaattg acacc 45 <210> 48 <211> 43 <212> DNA <213> Artificial Sequence <400> 48 taatacgact cactataggg actcccatgc tctgcacaat atc 43

Claims

1. A dsRNA for inducing RNA interference, characterized in that The sequence of the dsRNA is any one or more of SEQ ID NOs: 1-8 and their complementary sequences.

2. The dsRNA according to claim 1, wherein The dsRNA sequence is a combination of the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3, and their complementary sequences; a combination of the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, and their complementary sequences; a combination of the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, and their complementary sequences; a combination of the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 8, and their complementary sequences; a combination of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 8, and their complementary sequences; or a combination of the sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and their complementary sequences.

3. A modified dsRNA obtained by subjecting the dsRNA according to claim 1 or 2 to nucleotide modification.

4. A dsRNA combination for inducing RNA interference, characterized in that The dsRNA combination consists of any two of the dsRNAs according to claim 1.

5. The DNA encoding the dsRNA according to claim 1, characterized in that The DNA is a sequence shown in any one or more of SEQ ID NOs: 9-16 and a complementary sequence thereof.

6. The DNA according to claim 5, wherein the DNA is a combination of the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 11, and their complementary sequences; a combination of the sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11, and their complementary sequences; a combination of the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, and their complementary sequences; a combination of the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 16, and their complementary sequences; a combination of the sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 16, and their complementary sequences; or a combination of the sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, and their complementary sequences.

7. A modified DNA obtained by subjecting the DNA according to claim 5 or 6 to nucleotide modification.

8. A DNA combination encoding the dsRNA combination according to claim 4, characterized in that The DNA combination consists of any two of the DNAs described in claim 5.

9. A delivery vector for inducing RNA interference, characterized in that The delivery vector comprises the dsRNA of claim 1 or 2, or the modified dsRNA of claim 3, or the dsRNA combination of claim 4, or the DNA of claim 5 or 6, or the modified DNA of claim 7, or the DNA combination of claim 8.

10. The delivery vector according to claim 9, characterized in that The delivery vector is selected from viral vectors and non-viral vectors.

11. The delivery vector according to claim 10, characterized in that The non-viral vector is selected from the group consisting of liposomes, plasmid vectors, and phage vectors.

12. The delivery vector according to claim 10, characterized in that The viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector and a hybrid viral vector.

13. The delivery vector according to claim 10, characterized in that The viral vector is selected from adeno-associated viral vectors.

14. A kit for inducing RNA interference, characterized in that: The kit comprises the dsRNA of claim 1 or 2, or the modified dsRNA of claim 3, or the dsRNA combination of claim 4, or the DNA of claim 5 or 6, or the modified DNA of claim 7, or the DNA combination of claim 8, or the delivery vector of any one of claims 9 to 13.

15. A method for inhibiting Sf-Rhabdovirus from virus-contaminated organisms or cells, comprising: Obtain cells from virus-contaminated organisms or cell populations; After washing the above cells, the dsRNA according to claim 1 or 2, or the modified dsRNA according to claim 3, or the dsRNA combination according to claim 4, or the DNA according to claim 5 or 6, or the modified DNA according to claim 7, or the DNA combination according to claim 8 is introduced into the cells directly or via a delivery vector to induce RNA interference.

16. A method for obtaining an Sf-Rhabdovirus-free cell line from an organism or cell derived from a virus contamination, comprising: Obtain cells from virus-contaminated organisms or cell populations; After washing the cells, dsRNA having the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 8, and their complementary sequences, or modified dsRNA obtained by nucleotide modification using dsRNA having the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 8, and their complementary sequences, or DNA having the sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 16, and their complementary sequences, or modified DNA obtained by nucleotide modification using DNA having the sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 16, and their complementary sequences, is introduced into the cells directly or via a delivery vector to induce RNA interference; After washing and resuspending the above cells, the cells are separated into single cells or multiple cells; The isolated single cell or multiple cells are cultured and expanded in a medium suitable for cell growth and division to produce single-cell clones or multi-cell clones, thereby obtaining a virus-free cell line.

17. The method according to claim 16, characterized in that The method further comprises taking out a portion from the cell clone or the culture medium to detect the presence of the virus.

18. The method according to claim 17, characterized in that: The virus is an Sf-Rhabdovirus.

19. The method according to claim 15 or 16, characterized in that The cells are of insect origin.

20. The method according to claim 19, characterized in that The insects are Lepidoptera insects.

21. The method according to claim 19, wherein The insect is the fall armyworm.

22. The method according to claim 16, wherein The cell lines are derived from primary cells or cell lines that are contaminated with viruses.

23. The method according to claim 22, characterized in that The virus-contaminated cell line includes Sf21 or Sf9 cells, and the virus is an Sf-Rhabdovirus.

24. The method according to claim 15 or 16, characterized in that The introduction includes transfection or transduction.

25. The method according to claim 24, characterized in that The introduction is transfection.

26. The method according to claim 24, characterized in that The introduction is carried out by liposome transfection.

27. The method according to claim 17, wherein The detection comprises (a) RT-PCR; (b) RT-PCR and nested PCR; (c) quantitative RT-PCR; or (d) antibody-based detection technology.

28. The method of claim 16, wherein Methods for separation into single cells or multiple cells include limiting dilution cloning, cloning cells in soft agar and subsequent picking of cell colonies, cell sorting, laser capture microdissection (LCM), manual capture using a micropipette, microfluidics, or use of a micromanipulator.

29. The method of claim 28, wherein The method for separating into single cells or multiple cells is limiting dilution cloning.

30. The method of claim 29, wherein The plurality of cells is 2-20 cells.

31. The method of claim 29, wherein The plurality of cells is 3-10 cells.

32. The method of claim 29, wherein The plurality of cells is 4-6 cells.

33. The method of claim 29, wherein The plurality of cells is 5 cells.

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