CN115491377A - Nucleotide sequence for inducing RNA interference, reducing and eliminating virus pollution in cells and application - Google Patents

Abstract

The invention discloses a dsRNA sequence and a combination which can be used for reducing and eliminating Sf-rhabdovirus pollution in Sf cells, a corresponding siRNA sequence, a DNA sequence and a combination corresponding to the dsRNA sequence and the combination and the siRNA sequence, and also discloses a method for reducing and eliminating the Sf-rhabdovirus in the Sf cells by applying the nucleic acid sequence and an Sf-rhabdovirus-free cell line obtained by the method.

Description

Nucleotide sequence for inducing RNA interference, reducing and eliminating virus pollution in cells and application

Technical Field

The present invention relates to a nucleotide sequence and a combination for inducing RNA interference and reducing and eliminating virus pollution in cells, a method for obtaining a virus-free cell line by using the nucleotide sequence and the combination, and a continuous cell line which is free from pollution viruses and is obtained by using the method, wherein the virus-free cell line is derived from organisms or cell populations polluted by the viruses.

Background

Sf9 cell-based baculovirus expression systems have been widely used for expression and production of vaccines, proteins and genes. In 2014, a novel rhabdovirus exists in the cell line, so that the cell line has potential safety risks as a generation source of clinical treatment medicines.

Spodoptera frugiperda rhabdovirus (Sf-rhabdovirus) is a novel rhabdovirus that is present in Sf (Spodoptera frugiperda) cells (e.g., sf9, sf 21). The virus was discovered by researchers at the U.S. food and drug administration by degenerate PCR and Massively Parallel Sequencing (MPS). The Sf-rhabdovirus complete genome is 13584bp in length and mainly comprises 5 genes which respectively encode conserved proteins N (nucleocapsid), P (photoprotein), M (matrix), G (glycoprotein) and L (ploymerase), and an unknown gene encodes a non-conserved protein X, and the function of the protein is unknown. Sf-rhabdovirus belongs to single-stranded (-) RNA virus, and the replication mechanism of the virus is that RNA dependent RNA polymerase in virus particles transcribes RNA into mRNA, so as to express corresponding protein, and the protein is mainly proliferated in cytoplasm and released in a budding mode. Cannot be propagated alone, and can only be propagated in living cells.

There are two main strategies for controlling Sf-rhabdovirus: one is removal during sample production and purification; the second is the direct removal of Sf-rhabdovirus from Sf9 cells. The removal of Sf-rhabdovirus from the production and purification process can increase the production cost along with the increase of the production times of the medicine, and the Sf-rhabdovirus always exists in the production source cells, so that the hidden danger of pollution still exists, and the inevitable influence can be brought to the production. And the direct removal of Sf-rhabdovirus from Sf9 cells avoids virus pollution from the source, and can effectively control the Sf-rhabdovirus.

New insect cell lines free of exogenous viruses have been isolated and used as improved research tools and safer bio-manufacturing platforms. To completely eliminate Sf-rhabdovirus in Sf9 cells, gritecovist beck company treated a starting culture consisting of several cells with a nucleoside drug (e.g., 6-azauridine) and obtained a cell line free of Sf-rhabdovirus by further expansion culture (patent application No. CN 201680071306.3). In the practical screening, the patent application tries a method of co-inhibiting by using a plurality of medicaments, and the application of a plurality of antiviral medicaments can influence the cell metabolism and the biosynthesis process, aggravate cell damage and influence the capacity of cell recombination and virus production after screening. Although other studies show that a cell line with similar growth characteristics and better activity is obtained by screening without adding an antiviral drug (patent application No. CN 201910317758.0), the efficiency of obtaining a virus-free cell line by the method is very low.

In summary, the current method for inhibiting Sf-rhabdovirus in Sf9 cells mainly adopts nucleoside chemical drugs, but the inhibition mode belongs to broad-spectrum antiviral, and the inhibition of the virus may block the synthesis and transcription of the cell self genes and cause certain damage to the cells.

Thus, there remains a need in the art for methods of efficiently and stably obtaining and establishing virus-free cell lines from virus-contaminated organisms or cell populations, as well as cell lines that are free of contaminating viruses and that retain their original cellular biological properties and functions (e.g., cell viability, ability to produce recombinant viruses, etc.).

RNA interference (RNAi) is a phenomenon of gene silencing induced by double-stranded RNA (dsRNA) that, when introduced into a cell, causes degradation of an endogenous mRNA coding region, thereby inducing gene silencing. RNA interference was first discovered in caenorhabditis elegans in 1998, and then subsequently in eukaryotes such as fungi, drosophila, arabidopsis, insects, and mammals. The main mechanism of RNAi is that a single-stranded small RNA consisting of 20-30 nucleotides and Argonaute protein form a complex, and then a target RNA sequence is targeted through the Watson-Crick base pairing principle, so that the corresponding RNA is cut, and gene silencing is caused.

In animal cells, there are 3 kinds of small interfering RNAs, respectively siRNAs (small interfering RNAs), miRNAs (microRNAs), and piRNAs (PIWI-interfering RNAs), which differ in their production mechanism and effector function. In insect cells, the RNase-III enzyme Dicer-2 recognizes double-stranded RNA (dsRNA) in the cytoplasm and cleaves into 21nt size siRNA, where one strand binds to the Ago2 protein to form an RNA-induced silencing complex (RISC) and the other complementary strand is degraded. The Dicer-2 and RISC complex functions also require the involvement of multiple cofactors, including the dsRNA binding proteins Loqs-PD, ars2 and heat shock proteins. These proteins assist the biogenesis process of siRNA by stabilizing the RNA protein complex or assisting conformational changes. After the RNA silencing complex is formed, the 3' end of the RNA is methylated, the RISC eventually loaded with siRNA matures and recognizes the targeted RNA for cleavage according to the base pairing principle.

RNAi is currently widely used in the fields of virus resistance, gene regulation, gene therapy and the like. The interference modes of RNA are mainly divided into three types: chemically synthesizing siRNA, (2) transcribing and synthesizing long-chain dsRNA and specific or non-specific siRNA in vitro, and (3) expressing a vector by siRNA. The siRNA expression vector is characterized in that a DNA template sequence corresponding to siRNA is inserted into an expression plasmid, transcription of RNA is started after the plasmid is transferred into a cell, a hairpin structure of shRNA (small hairpin RNA) is formed, and then the siRNA is cut into the siRNA under the action of Dicer enzyme.

The chemical synthesis approach is to customize short siRNA molecules, each directed to a target, and commercial companies are primarily designed and customized for mammalian cells. The siRNA expression vectors currently in commercial use are also directed to in vivo expression in mammalian cells. Aiming at Sf9 cells, plasmids need to be designed and constructed by self, each plasmid targets one target point, a large amount of preliminary research is needed to determine effective plasmids, the time is long, and therefore, proper siRNA needs to be designed to solve the problems. The long-chain dsRNA is sheared into a plurality of small siRNA molecules after being transferred into cells, and the siRNA can act on a plurality of targets, so that the RNA interference efficiency is improved.

The present invention adopts RNA interference (RNAi) mode, especially long chain dsRNA to inhibit Sf-rhabdovirus replication, and obtains Sf-rhabdovirus-free cell line (SF-RVN) via separating and culturing single cell clone or multiple cell clone. The RNAi method not only avoids the addition of exogenous drugs, but also reduces the risk, and moreover, no report is made on the method for inhibiting Sf-rhabdovirus in Sf9 cells by using RNAi.

Disclosure of Invention

The invention provides a nucleic acid molecule or a combination thereof, which induces RNA interference and reduces and eliminates viral contamination in cells, and comprises deoxyribonucleic acid (RNA) or a combination thereof and ribonucleic acid (DNA) or a combination thereof.

The invention provides dsRNA, the sequence of which is SEQ ID NO:1-8, or a combination thereof.

In one embodiment of the invention, the one dsRNA sequence is: the amino acid sequence of SEQ ID NO:1 and SEQ ID NO:3 in combination; SEQ ID NO:2 and SEQ ID NO:3 combined sequences; the amino acid sequence of SEQ ID NO:3 and SEQ ID NO:4 in combination; SEQ ID NO:3 and SEQ ID NO:8 in combination; SEQ ID NO: 1. SEQ ID NO: 2. the amino acid sequence of SEQ ID NO: 3. SEQ ID NO:4 and SEQ ID NO:8 in combination; or SEQ ID NO: 5. the amino acid sequence of SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8 in combination.

In certain embodiments, the dsRNA is a modified dsRNA obtained by modifying a nucleotide of the above sequence.

The invention also provides a dsRNA combination consisting of any two or more of the above dsrnas.

In one embodiment of the invention, the dsRNA combination is: SEQ ID NO:1 and SEQ ID NO:3, a combination of dsrnas set forth herein; SEQ ID NO:2 and SEQ ID NO:3, a combination of dsrnas set forth herein; SEQ ID NO:3 and SEQ ID NO:4, a combination of dsrnas set forth herein; SEQ ID NO:3 and SEQ ID NO:8, a combination of dsrnas set forth herein; SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2. the amino acid sequence of SEQ ID NO: 3. SEQ ID NO:4 and SEQ ID NO:8, a combination of dsrnas set forth herein; or SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8 in combination with a dsRNA.

The invention also provides siRNA molecules obtained by cleaving the aforementioned dsRNA or combination of dsRNA.

In another embodiment of the present invention, the siRNA may consist of SEQ ID NO:1-8 or a combination thereof, preferably 16-25, more preferably 18-21 contiguous nucleotides.

The invention also provides a DNA sequence for coding the dsRNA. In one embodiment of the present invention, a DNA sequence of the present invention encoding the above dsRNA is SEQ ID NO:9-16, or a combination thereof.

In certain embodiments of the invention, the DNA sequence encoding the dsRNA is: SEQ ID NO:9 and SEQ ID NO:11 in combination; the amino acid sequence of SEQ ID NO:10 and SEQ ID NO:11 in combination; the amino acid sequence of SEQ ID NO:11 and SEQ ID NO:12 in combination; SEQ ID NO:11 and SEQ ID NO:16 in combination; the amino acid sequence of SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. the amino acid sequence of SEQ ID NO:12 and SEQ ID NO:16 in combination; or SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO:16 in combination.

In some embodiments, the DNA is a modified DNA obtained by modifying a nucleotide of the above sequence.

The invention also provides a DNA combination encoding a dsRNA combination consisting of the amino acid sequence of SEQ ID NO: 9-16.

In certain embodiments of the invention, the combination of DNA encoding the combination of dsRNA is: the amino acid sequence of SEQ ID NO:9 and SEQ ID NO:11 in combination with the DNA shown in fig. 11; SEQ ID NO:10 and SEQ ID NO:11 in combination with the DNA shown in fig. 11; SEQ ID NO:11 and SEQ ID NO:12 in the presence of a DNA fragment; SEQ ID NO:11 and SEQ ID NO:16 in combination with a DNA shown in seq id no; the amino acid sequence of SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12 and SEQ ID NO:16 in combination with a DNA shown in seq id no; or SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO:16, or a combination of the DNAs shown in.

In certain embodiments of the invention, DNA encoding the above-described siRNA is provided.

The invention also provides a delivery vector comprising the above-described nucleic acid molecules (including RNA or DNA), or a combination thereof. The delivery vector is selected from the group consisting of viral vectors and non-viral vectors. Preferably, the non-viral vector is selected from the group consisting of a liposome, a plasmid vector, a phage vector. Preferably, 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. More preferably, the viral vector is selected from adeno-associated viral vectors. The purpose of the delivery vector is to deliver the nucleic acid molecule described above into a cell to induce RNA interference.

The invention also provides a kit, which comprises the RNA (including dsRNA, siRNA and the like) or the combination thereof, the DNA or the combination thereof and a delivery carrier.

The invention also provides a method for establishing and obtaining a virus-free cell line, wherein the nucleic acid molecules (including RNA or DNA) or the combination thereof and the delivery vector containing the nucleic acid molecules (including RNA or DNA) or the combination thereof are utilized to reduce and eliminate virus pollution in cells through an RNA interference technology.

The specific method comprises the following steps:

(1) Obtaining cells from a virally contaminated organism or population of cells;

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

(3) After washing and resuspending the cells, separating the cells into single cells or multiple cells;

(4) The isolated single cell or plurality of cells are expanded in culture in a medium suitable for cell growth and division to produce single cell clones or multi-cell clones, thereby obtaining virus-free cell lines.

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

In one aspect, the virus contaminating the organism or cell population described in the present invention is an Sf-rhabdovirus. In another aspect, the virally contaminated organisms of the invention are insects, preferably Spodoptera frugiperda (Sf); the virus-contaminated cell population is an established cell line or cell strain, such as a commercially available or marketed cell line, preferably an insect cell line, more preferably an Sf cell line (e.g., sf9 cell line, sf21 cell line, etc.).

Thus, the cell line of the invention may be derived directly or indirectly from spodoptera frugiperda, or may be derived from a commercially available or commercial Sf9 or Sf21 cell line.

Further, the present invention provides in another aspect a virus-free Sf cell line (referred to herein as SF-RVN cell line) derived from a commercially available or commercial Sf9 or Sf21 cell line and established by the above method.

Compared with the prior art, the invention has the following beneficial technical effects:

at present, a method for inhibiting Sf-rhabdovirus in Sf9 cells mainly adopts nucleoside chemical drugs, but the inhibition mode belongs to broad-spectrum antiviral, and the nucleoside drugs are added into the cultured cells, so that the synthesis, transcription and metabolism of the genes of the cells can be hindered while the virus is inhibited, and certain damage is caused to the cells. The invention provides a nucleic acid molecule or a combination thereof for reducing and eliminating Sf-rhabdovirus pollution in Sf9 cells, specifically inhibits the Sf-rhabdovirus by adopting an RNA interference mode, reduces damage to the cells, can effectively inhibit the replication of the virus, and can obtain a stable virus-free cell line.

Drawings

FIG. 1. Specific DNA templates obtained by PCR. PCR was performed on cDNA obtained by reverse transcription of Sf-rhabdovirus mRNA using 8 specific primers designed, and the products are shown in the figure. M represents Marker (Trans 2K DNA Marker, holotype gold, BM 101-01), 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.

FIG. 2. Specific T7 DNA templates were obtained by PCR. PCR is carried out on 8 specific DNA templates by using 8 designed specific T7 primers, and the products are shown in the figure. M represents Marker (Trans 2K DNA Marker, all-formula gold, BM 101-01), 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.

FIG. 3 dsRNA is obtained by in vitro transcription of specific T7 DNA template. Taking DNA as a template, transcribing single-stranded RNA in vitro, combining two complementary single-stranded RNAs into a long-chain dsRNA through heating (denaturation) and annealing, and then purifying to obtain the dsRNA for transfecting cells. (A) The dsRNA-L3-T7 DNA template is transcribed in vitro to obtain the corresponding dsRNA. M represents Marker (Trans 2K DNA Marker, all-round gold, BM 101-01), 1 represents L3-dsRNA (before denaturation), 2 represents L3-dsRNA (after annealing), and 3 represents L3-dsRNA (after purification). (B) in vitro transcription of the T7 DNA template to obtain the corresponding dsRNA. M represents Marker (Trans 2K DNA Marker, all-round gold, BM 101-01), 1 represents L1-dsRNA (after purification), 2 represents L2-dsRNA (after purification), and 3 represents L4-dsRNA (after purification). (C) in vitro transcription of the T7 DNA template to obtain the corresponding dsRNA. M represents Marker (Trans 2K DNA Marker, all-round gold, BM 101-01), 1 represents N-dsRNA (after purification), 2 represents P-dsRNA (after purification), 3 represents M-dsRNA (after purification), and 4 represents G-dsRNA (after purification).

Detailed Description

To facilitate an understanding of the various embodiments of the disclosure, the following explanation of specific terms is provided.

Cell line: refers to a cell population expanded from one or several common ancestor cells, including, but not limited to, a cell population expanded from a single isolated cell.

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

Derived from an organism: means obtained directly or indirectly from an organism. The cells may be derived directly from the organism, e.g. by obtaining a tissue or organ from the organism and then lysing the tissue or organ to obtain primary cells. The cells may also be indirectly derived from the organism, for example, by isolating single cells from a cell line derived from the organism and then expanding the isolated single cells to establish and obtain the cell line.

The organism of the invention may be an insect, in particular a lepidopteran insect. Lepidopteran insects refer to any member of the order lepidopteran, including butterflies, moths, etc., whose adults have four broad or lanceolated wings, typically covered with tiny overlapping and brightly colored scales, and whose larvae are caterpillars. For example, lepidopteran insects include, but are not limited to, spodoptera frugiperda, cabbage looper, or bombyx mori.

Single cell clones are defined as cell populations that are formed by the expansion of individual cell cultures. Polyclonal cells refer to a population of cells formed by the expansion of a plurality of cells in culture.

dsRNA (double-stranded RNA) or double-stranded RNA refers to a double-stranded RNA molecule consisting of two RNA sequences that are complementary in opposite directions, which, upon entering a cell, can be cleaved into siRNA, thereby inducing RNA interference.

siRNA (small interfering RNA), also known as small interfering RNA or short interfering RNA, is a type of double-stranded RNA of length typically 19-25 base pairs, mainly involved in RNA interference, causing degradation at the mRNA level, resulting in hindered protein translation.

"comprising" or "including" is synonymous and is an open-ended term that does not exclude the presence of other unrecited components, elements, steps, or the like. For example, a composition "comprising" components a, B and C may consist of components a, B and C; alternatively, the composition may comprise not only components a, B and C, but also one or more other components.

The terms "detecting the presence of a virus", "detecting the presence of Sf-rhabdovirus" and related terms are used broadly in this specification. Those skilled in the art will appreciate that there are many detection techniques known in the art that can be used with 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 observation of cytopathic effect (CPE), bioinformatics techniques such as BLAST searches, and the like.

The invention provides a nucleic acid molecule or combination thereof, comprising deoxyribonucleic acid (RNA) or a combination thereof and ribonucleic acid (DNA) or a combination thereof, which can inhibit Sf-rhabdovirus, or can reduce and eliminate Sf-rhabdovirus contamination in cell lines.

In one embodiment of the invention, the RNA comprises dsRNA and siRNA.

In another embodiment of the invention, the sequence of one dsRNA is SEQ ID NO:1-8, or a combination thereof. In one embodiment of the present invention, the dsRNA sequence refers to the following sequence in a dsRNA molecule: i.e. consisting of SEQ ID NO:1-8 in combination (e.g., by covalent linkage).

In one embodiment of the invention, the sequence of the dsRNA is preferably SEQ ID NO:3, and (b) is shown in the specification.

In one embodiment of the present invention, the dsRNA sequence is: SEQ ID NO:1 and SEQ ID NO:3 combined sequences; the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:3 combined sequences; SEQ ID NO:3 and SEQ ID NO:4 in combination; SEQ ID NO:3 and SEQ ID NO:8 in combination; the amino acid sequence of SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2. the amino acid sequence of SEQ ID NO: 3. SEQ ID NO:4 and SEQ ID NO:8 in combination; or SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8 in combination.

In one embodiment of the invention, said one dsRNA sequence is preferably SEQ ID NO:3 with other sequences.

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

The invention also relates to a combination of dsrnas, that is, a combination of any two or more of the dsrnas disclosed herein. In a particular embodiment of the invention, the dsRNA combination refers to any two or more dsrnas used or applied in combination or in combination in the same use or method or kit.

In one embodiment of the invention, the dsRNA is a combination of: SEQ ID NO:1 and SEQ ID NO:3, a combination of dsrnas set forth herein; the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:3, a combination of dsrnas set forth herein; SEQ ID NO:3 and SEQ ID NO:4, a combination of dsrnas set forth herein; SEQ ID NO:3 and SEQ ID NO:8, a combination of dsrnas set forth herein; the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. the amino acid sequence of SEQ ID NO: 3. SEQ ID NO:4 and SEQ ID NO:8, a combination of dsrnas set forth herein; or SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8 in combination with a dsRNA.

In one embodiment of the invention, a combination of dsrnas is preferably SEQ ID NO:3 with other dsrnas of the invention.

The invention also provides siRNA molecules obtained by cleaving the above dsRNA or dsRNA combination.

In another embodiment of the present invention, the siRNA may consist of SEQ ID NO:1-8 or a combination thereof, preferably 16-25, more preferably 18-21 contiguous nucleotides.

The invention also provides a DNA for coding the dsRNA.

In certain embodiments of the invention, the DNA sequence encoding the dsRNA is SEQ ID NO:9-16, or a combination thereof. In one embodiment of the present invention, the DNA sequence encoding the dsRNA is the following sequence in a DNA molecule: i.e. consisting of SEQ ID NO:9-16, in combination (e.g., by covalent linkage).

In one embodiment of the invention, a DNA sequence encoding the above dsRNA is preferably SEQ ID NO:11, and (b) is the sequence shown in the specification.

In one embodiment of the present invention, a DNA sequence encoding the above dsRNA is: the amino acid sequence of SEQ ID NO:9 and SEQ ID NO:11 in combination; SEQ ID NO:10 and SEQ ID NO:11 in combination; SEQ ID NO:11 and SEQ ID NO:12 in combination; SEQ ID NO:11 and SEQ ID NO:16 in combination; SEQ ID NO: 9. the amino acid sequence of SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 11. SEQ ID NO:12 and SEQ ID NO:16 in combination; or SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO:16 in combination.

In one embodiment of the invention, the DNA sequence encoding the above dsRNA is preferably SEQ ID NO:11 with other sequences.

In some embodiments, the DNA is a modified DNA obtained by modifying a nucleotide of the above sequence. Such modifications include, but are not limited to, chemical modifications that improve the stability of the DNA in vivo, and the specific types and means of modification are well known in the art.

The invention also relates to combinations of DNA, i.e., any two or more of the DNA disclosed herein. In a specific embodiment of the invention, the combination of DNA refers to any two or more DNAs used or applied in combination or combination in the same use or method or kit.

In certain embodiments of the invention, combinations of the above DNAs are provided. Wherein the combination is: the amino acid sequence of SEQ ID NO:9 and SEQ ID NO:11 in combination with the DNA shown in fig. 11; the amino acid sequence of SEQ ID NO:10 and SEQ ID NO:11 in the above-mentioned DNA combinations; SEQ ID NO:11 and SEQ ID NO:12 in the presence of a DNA fragment; SEQ ID NO:11 and SEQ ID NO:16 in combination with a DNA shown in seq id no; the amino acid sequence of SEQ ID NO: 9. the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 11. the amino acid sequence of SEQ ID NO:12 and SEQ ID NO:16 in combination with a DNA shown in seq id no; or SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO:16, or a combination of the DNAs shown in.

In one embodiment of the invention, the combination of DNA is preferably SEQ ID NO:11 with other DNAs of the present invention.

In certain embodiments of the invention, the DNA is a DNA encoding an siRNA as described above.

The invention also provides a delivery vector comprising the above-described nucleic acid molecules (including RNA or DNA), or a combination thereof, for inducing gene silencing or RNA interference. The delivery vector is selected from the group consisting of viral vectors and non-viral vectors. Preferably, the non-viral vector is selected from the group consisting of a liposome, a plasmid vector, a phage vector. Preferably, 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. More preferably, the viral vector is selected from adeno-associated viral vectors. In a particular embodiment of the invention, the delivery vehicle comprises a dsRNA or a combination of dsRNA or a DNA or a combination of DNA as described above. In another embodiment of the invention, different dsRNA or DNA molecules in the dsRNA or DNA are in the same delivery vector, in the same expression cassette. In another embodiment of the invention, the different dsRNA or DNA molecules of a dsRNA combination or a DNA combination may be in the same expression cassette or in different expression cassettes. In another embodiment of the invention, the different dsRNA or DNA molecules of a dsRNA combination or a DNA combination may be in the same delivery vehicle or in different delivery vehicles.

In another aspect, the invention provides a kit useful for inducing RNA interference. In a specific embodiment of the invention, the kit comprises the dsRNA or combination thereof, DNA or combination thereof, siRNA and/or a delivery vehicle of the invention described above.

In another aspect, the invention provides a method for establishing and obtaining Sf-free rhabdovirus cell lines using the above-described nucleic acid molecules (including RNA or DNA) or combinations thereof, delivery vectors comprising the above-described nucleic acid molecules (including RNA or DNA) or combinations thereof, RNA interference techniques to reduce and eventually eliminate contaminating Sf-rhabdovirus from cells.

Certain exemplary methods for obtaining a virus-deficient cell line comprise: obtaining cells from a virally contaminated organism or population of cells; after washing the above cells, introducing the dsRNA or combination thereof, or DNA or 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 re-suspending the introduced cells, and separating the cells into single cells or a plurality of cells; the isolated single cell or multiple cells are cultured and expanded in a culture medium suitable for cell growth and division to form single cell clones or multiple cell clones, so as to obtain the virus-free cell line.

In certain embodiments, the above method for obtaining a cell line lacking a virus further comprises removing a portion from the above cell clone or the culture medium to detect the presence of a virus.

In certain embodiments, the virus contaminating an organism or cell population described herein is an Sf-rhabdovirus. In some embodiments, the virally-contaminated organisms described herein are insects, preferably spodoptera frugiperda; the virus-contaminated cell population is an established cell line or cell strain, e.g., a commercially available or marketed cell line, preferably an insect cell line, more preferably an Sf9 or Sf21 cell line.

In certain embodiments, techniques for RNA interference using nucleic acid molecules are well known in the art, and methods for delivering nucleic acid molecules in RNA interference primarily include delivery by non-viral vectors, including but not limited to liposomes, plasmid vectors, phage vectors, or bacterial vectors, or/and by viral vectors; viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors or hybrid viral vectors.

In certain embodiments, a single cell refers to only 1 cell; by plurality of cells is meant a cell containing several cells, including, but not limited to, 2-20 cells, preferably 3-10 cells, preferably 4-6 cells, more preferably 5 cells.

In certain embodiments, methods of isolating into a single cell 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 micromanipulators.

In certain embodiments, methods for detecting the presence of viruses, including but not limited to Sf-rhabdoviruses, are well known in the art and include, but are not limited to, RT-PCR, nested PCR, RT-PCR in combination with nested PCR, or RT-QPCR.

In certain embodiments, after a cell clone that is to be tested for the absence of virus is expanded in normal medium for a period of time, a portion of the expanded cells and supernatant culture medium are removed to again verify complete removal of the virus.

In certain embodiments, the established virus-free cell lines are derived from primary cells contaminated with viruses, including but not limited to Sf-rhabdoviruses. In certain embodiments, the primary cells contaminated with a virus are from a virus-contaminated organism (including but not limited to Spodoptera frugiperda).

In other embodiments, the established virus-free cell lines are derived from a population of cells (including but not limited to commercially available or commercial cell lines) contaminated with viruses (including but not limited to Sf-rhabdoviruses). 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.

It will be appreciated by those skilled in the art that materials and conditions suitable for growth and incubation of a particular cell type are known in the art, as are sources of access. Such sources include, but are not limited to, cell culture manuals, commercial cell banks, or media suppliers. Suitable cell culture conditions can also be readily determined using methods known in the art.

The present invention is further illustrated by the following specific examples. It is to be understood that the following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention.

The techniques used in the following examples, including biological techniques such as gene sequencing, PCR amplification and detection, cell transfection, cell culture, and the like, are conventional techniques known to those skilled in the art unless otherwise specified; the instruments, reagents, etc. used, unless otherwise specifically noted in this specification, are publicly available to those skilled in the art.

Example 1 DNA template preparation

8 specific targeting Sf-rhabdovirus genomes of 500-900bp are designed and obtained according to conserved sequences in the Sf-rhabdovirus genomes (GenBank access number KF 947078.1), and long-chain dsRNA sequences of Sf21 or Sf9 cell genomes are not targeted, wherein specific sequences are shown in a table 1, and corresponding DNA sequences are shown in a table 2.

TABLE 1 dsRNA sequences

TABLE 2 dsRNA corresponding DNA

Total mRNA of Sf9 cells and Sf-rhabdovirus was extracted using RNeasy Plus Mini kit (Qiagen, 74134), followed by reverse transcription of mRNA to synthesize cDNA (kit used was

III First-Strand Synthesis System for RT-PCR，Invitrogen，18080051)。

8 pairs of specific primers (specific sequences are shown in Table 3) which are designed and synthesized are used for carrying out PCR amplification on the cDNA (a kit is Pyrobest DNA polymerase, takara, R005A), 8 specific DNA templates are amplified (the reaction program is 94 ℃ for 3min, 3094 ℃ for 94 ℃,60 ℃ for 30s, and 72 ℃ for 172 min, 8 ℃ for +/-infinity), and the PCR amplification results are shown in a figure 1, and the DNA templates are respectively recovered.

TABLE 3 dsRNA primer design

8 specific DNA templates (dsRNA-N, dsRNA-P, dsRNA-M, dsRNA-G, dsRNA-L1, dsRNA-L2, dsRNA-L3 and dsRNA-L4) are respectively connected to a T vector for sequencing verification. Sequencing results showed that the specific DNA template synthesized matched the sequence on the Sf-rhabdovirus genome.

Using 8 pairs of specific T7 primers (the specific sequence is shown in Table 4) which are designed and synthesized to respectively carry out PCR amplification on the recovery products of the specific DNA templates (the kit is Pyrobest DNA polymerase, takara, R005A), respectively amplifying 8 specific T7 DNA templates (the reaction program is 94 ℃ for 3min 30cycle for 94 ℃ 30s,58 ℃ 30s,72 ℃ for 110 min, 8 ℃ and +/-infinity), and the PCR amplification result is shown in a figure 2, recovering the products and using for synthesizing dsRNA.

TABLE 4 dsRNA T7 primer design

EXAMPLE 2 in vitro transcription Synthesis of dsRNA

In vitro Synthesis of dsRNA: comprises in vitro transcription, denaturation annealing, impurity removal of DNA and ssRNA, purification, nucleic acid gel electrophoresis, etc., as detailed in MEGAscript ^TM RNAi kit instructions (Invitrogen, AM 1626) resulted in 8 high-concentration, relatively single bands of dsRNA available for transfection (table 5, fig. 3).

Table 5 concentrations of 8 dsRNA synthesized

Example 3 Sf-Rhabdoviral assay

To verify the presence of Sf-rhabdovirus in the cells and supernatant culture, RNA was extracted from transfected cell supernatants and cells and supernatants at different passage levels and RT-PCR was used to detect Sf-rhabdovirus (see: ma et al, J.Virol.88:6576-85, 2014). In addition, in order to detect the presence of Sf-rhabdovirus more accurately and precisely, a set of primers and probes were designed using Primer design software (Primer Premier 6.25) based on the Sf-rhabdovirus L sequence (Genbank: KF 947078.1), and the one-step quantitative RT-PCR (RT-QPCR) method was used to quantitatively detect the Sf-rhabdovirus content. Firstly, RNA in cells and culture supernatant is extracted, then a TaqMan RNA-to CT 1-Step kit (ABI/Thermo, 4392938) is adopted, and one-Step RT-QPCR is carried out according to the operation instruction of the kit of the manufacturer to detect the content of the Sf-rhabdovirus.

EXAMPLE 4 dsRNA transfected cells

Sf9 cells were cultured in serum-free medium (ESF AF, expression Systems) and first 8 synthetic dsRNA and transfection reagent (Cellffectin) were selected ^TM II Reagent, invitrogen, 10362100), see Cellffectin ^TM II Reagent kit (Invitrogen, 10362100) instructions, sf-rhabdovirus content in the culture supernatant was assayed 2 days after cell transfection, as described in example 3. Each of the 8 dsrnas significantly reduced Sf-rhabdovirus content relative to untransfected Sf9 cells when tested by RT-PCR (results not shown). The results of RT-QPCR assays are shown in Table 6, where + indicates no additionPositive control for dsRNA.

TABLE 6 Sf-rhabdovirus content in supernatants of 8 dsRNA alone transfected cells

The result shows that the designed and synthesized 8 dsRNA has obvious inhibiting effect on Sf-rhabdovirus after independently transfecting Sf9 cells, and the content of the Sf-rhabdovirus in cell supernatant is obviously reduced, wherein the inhibiting effect of the M-dsRNA is most obvious and is reduced by 67.8%.

Then, transfection was performed by a combination of multiple dsrnas, and the Sf-rhabdovirus content in the supernatant medium was assayed to verify the inhibitory effect of the dsRNA combination on Sf-rhabdovirus, as described in example 3. The results of RT-QPCR assays are shown in Table 7, where, + represents a positive control without dsRNA addition.

TABLE 7 Sf-rhabdovirus content in supernatants of different dsRNA-transfected cells

The results show that compared with single dsRNA, the inhibition effect of different dsRNA combinations on Sf-rhabdovirus is further improved, wherein M + P is reduced by 76%, and M + N + P + G + L4 is reduced by 75%. And finally, selecting two modes of M + P and M + N + P + G + L4 for further research and verification.

EXAMPLE 5 screening of Sf-Virus-free cell clones and identification of Sf-rhabdoviruses

And transfecting Sf9 cells by respectively adopting two combination modes of M + P and M + N + P + G + L4, and after washing the cells, transfecting. After 2 days of transfection, the cells were washed, resuspended, separated into single cells (1 cell/well) or multiple cells (5 cells/well) by limiting dilution, and the cells were subjected to expansion culture in normal medium to form single-cell clones and multi-cell clones, and sampled for Sf-rhabdovirus detection, as described in example 3.

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

Two of the screened Sf-rhabdovirus-free cells, 38C3 and 38B4 (both from 5 cell/well polyclonals), were selected for further validation, 38C3 and 38B4 cells were expanded, and the cells were transferred to a T25 square flask defined as passage P0, and cells from different generations and Sf-rhabdovirus in the supernatant medium were detected as described in example 3. The results of the Sf-rhabdovirus detection are shown in Table 8.

Table 8 detection of Sf-rhabdovirus of selected cells

As a result, it was found that Sf-rhabdovirus was efficiently inhibited by using the designed long-chain dsRNA, and that stable Sf-rhabdovirus-free cell lines (SF-RVN cells) were established, which was based on the results of RT-PCR and RT-QPCR with high sensitivity. The above methods demonstrated that no Sf-rhabdovirus was detected in both SF-RVN cells (selected Sf-rhabdovirus-free cell lines) and supernatant media after at least 17 passages.

Through functional studies on the screened SF-RVN cells, the SF-RVN cells and Sf9 cells are found to be consistent in basic cell growth characteristics such as cell viability, cell diameter and cell agglomeration rate (results are not shown). SF-RVN cells and Sf9 cells are basically equivalent in baculovirus passage stability, recombinant AAV virus yield and the like (results are not shown), which indicates that the activity of Sf9 cells is not influenced by the method of the invention.

The results show that the screened SF-RVN cell can completely replace Sf9 cell and becomes a safe production cell of protein, virus and vaccine.

Sequence listing

<110> Shutaishen (Beijing) biopharmaceutical corporation; beijing Sanokui-Yi Biotechnology Limited liability company

<120> a nucleotide sequence for inducing RNA interference, reducing and eliminating virus pollution in cells and application

<160> 48

<210> 1

<211> 524

<212> RNA

<213> Artificial Sequence (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 (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

auuggacgaa cuuauagaca ucugucuucu ggggccuaag acucuaauug cuaccuuacg 420

aauggcgaua accgccuaua ccgcugcauu agccagaaau gccaagucca ccaucucaga 480

uauuacuacc gcaucagcag auuugauggu caucacucag augauacagu cccagcagga 540

aucuuuccaa ucaucauuag agcaucucuc ucaugcuugg aauaacgu 588

<210> 3

<211> 587

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

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 (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 (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 (Artificial Sequence)

<400> 6

uaugaaggag gcucguggaa agauucuaaa uuccggcaug aaauuguuaa agauuuagag 60

aaaaaagccu cagaucuaaa uuuacauccu caacuuagag uuagagaaca acuguuagau 120

ucaguuuuug aacgaaaucu aaacgccuuc acccaacugu augggcuaua ucgcauaugg 180

ggucacccaa cucuggaucc auuacuuggg acaauagccc ucaaagaauu gggaacaaca 240

ccaagauugu accuaucaca ccaagcucag gagauuaaca acaaguuuaa ggaagaguuc 300

auaaaaagau auuuaaauag acauaaggag uggccggaau uagauguauc gaaauuacca 360

agacauaaca ucauucgagu ccauuaugag aagaaauuac aauuuccuuc uaaauccaga 420

caauauagga gaucucaucu cuccuuggua gaauucaaag agguauuccc uguugauccu 480

aaauuugauc uuauugaauu uauugaugau aaauccaucu ccuuagguuu cccagaucuc 540

cuuaacgaga ucuauagaaa caagaguauc gggaauucac uagcaagauc cuuauugcuu 600

aauuuccucu ccucugacau uucagacccc caagaauuuc ugaagaauau agauaccuca 660

ggguuuccuc cugaagagau uuguguuggg guacacgaaa 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 (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 (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 (Artificial Sequence)

<400> 9

ggagtgttga tacatgtcgg gagatcagtt taaatctgaa gttacctggt gaaatatggc 60

aactggccca tcaagaaacc atcttcaaca gatttcttac attttacgct actgggtatg 120

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 480

tacatgtaga ggatgatatc aacgctctaa ctcagattac aaaa 524

<210> 10

<211> 588

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcctctcaag agaaactatc aactgattta ctctataatg 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 ggaaaatata 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 (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 aacaactgca 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 (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 (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 aattaagttt atttcttaag 600

gaaggagatg aaatgctaac tttagcagga aatcaaggat atgatctaat ttatacttta 660

gaatcttcct gtacttcccg at 682

<210> 14

<211> 855

<212> DNA

<213> Artificial Sequence (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

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 (Artificial Sequence)

<400> 15

ccaagtggaa caccaatatg agagccccag acacacagcc attttaccac 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 (Artificial Sequence)

<400> 16

attccaatgc aatattagaa gaccctatct ccaaaagaac tcattttatc aatcgttaga 60

tcgaaataga 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

ac 602

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

caattcatac atcccaataa ccc 23

<210> 18

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atcgggaagt acaggaagat tctaaag 27

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tatgaaggag gctcgtggaa ag 22

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tttctccaaa actaactcgt catcc 25

<210> 21

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ccaagtggaa caccaatatg agag 24

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ggagataggg tcttctaata ttgca 25

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

attccaatgc aatattagaa gaccc 25

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gtggagttgc ctgaagtaga agag 24

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ggagtgttga tacatgtcgg gag 23

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ttttgtaatc tgagttagag cgttg 25

<210> 27

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tcctctcaag agaaactatc aactg 25

<210> 28

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

acgttattcc aagcatgaga gagat 25

<210> 29

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gacatcaatc ataactttcc tcgg 24

<210> 30

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gttcttgggg cttgtttggt tg 22

<210> 31

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ttatggcaag agagaattga cacc 24

<210> 32

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

ctcccatgct ctgcacaata tc 22

<210> 33

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

taatacgact cactataggg acaattcata catcccaata accc 44

<210> 34

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

taatacgact cactataggg aatcgggaag tacaggaaga ttctaaag 48

<210> 35

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

taatacgact cactataggg atatgaagga ggctcgtgga aag 43

<210> 36

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

taatacgact cactataggg atttctccaa aactaactcg tcatcc 46

<210> 37

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

taatacgact cactataggg accaagtgga acaccaatat gagag 45

<210> 38

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

taatacgact cactataggg aggagatagg gtcttctaat attgca 46

<210> 39

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

taatacgact cactataggg aattccaatg caatattaga agaccc 46

<210> 40

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

taatacgact cactataggg agtggagttg cctgaagtag aagag 45

<210> 41

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

taatacgact cactataggg aggagtgttg atacatgtcg ggag 44

<210> 42

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

taatacgact cactataggg attttgtaat ctgagttaga gcgttg 46

<210> 43

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

taatacgact cactataggg atcctctcaa gagaaactat caactg 46

<210> 44

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

taatacgact cactataggg aacgttattc caagcatgag agagat 46

<210> 45

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

taatacgact cactataggg agacatcaat cataactttc ctcgg 45

<210> 46

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

taatacgact cactataggg agttcttggg gcttgtttgg ttg 43

<210> 47

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

taatacgact cactataggg attatggcaa gagagaattg acacc 45

<210> 48

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

taatacgact cactataggg actcccatgc tctgcacaat atc 43

Claims (33)

1. A dsRNA for inducing RNA interference, wherein the sequence of said dsRNA is SEQ ID NO:1-8, or a combination thereof.

2. The dsRNA of claim 1, wherein said sequence is SEQ ID NO:1 and SEQ ID NO:3 combined sequences; SEQ ID NO:2 and SEQ ID NO:3 in combination; the amino acid sequence of SEQ ID NO:3 and SEQ ID NO:4 in combination; SEQ ID NO:3 and SEQ ID NO:8 in combination; the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO:4 and SEQ ID NO: 8; or SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO: 8.

3. A modified dsRNA obtained by nucleotide modification of the dsRNA of claim 1 or 2.

4. A dsRNA combination for inducing RNA interference wherein said dsRNA combination consists of any two or more of the dsrnas of claim 1.

5. The dsRNA combination of claim 4, wherein said dsRNA combination is SEQ ID NO:1 and SEQ ID NO:3, a combination of dsrnas set forth herein; SEQ ID NO:2 and SEQ ID NO:3, a combination of dsrnas set forth herein; the amino acid sequence of SEQ ID NO:3 and SEQ ID NO:4, a combination of dsrnas set forth herein; SEQ ID NO:3 and SEQ ID NO:8, a combination of dsrnas set forth herein; SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO:4 and SEQ ID NO:8, a combination of dsrnas set forth herein; or SEQ ID NO: 5. the amino acid sequence of SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8, or a combination of dsrnas as set forth in seq id no.

6. siRNA obtained by cleavage of the dsRNA or dsRNA combination of any one of claims 1-5.

7. The siRNA of claim 6, whose sequence consists of SEQ ID NO:1-8 or a combination thereof, preferably 16-25, more preferably 18-21 contiguous nucleotides.

8. A DNA sequence encoding the dsRNA of claim 1, wherein the DNA sequence is SEQ ID NO:9-16, or a combination thereof.

9. The DNA sequence of claim 8, wherein the DNA sequence is SEQ ID NO:9 and SEQ ID NO:11 in combination; the amino acid sequence of SEQ ID NO:10 and SEQ ID NO:11 in combination; the amino acid sequence of SEQ ID NO:11 and SEQ ID NO:12 in combination; SEQ ID NO:11 and SEQ ID NO:16 in combination; SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. the amino acid sequence of SEQ ID NO:12 and SEQ ID NO:16 in combination; or SEQ ID NO: 13. SEQ ID NO: 14. the amino acid sequence of SEQ ID NO:15 and SEQ ID NO:16 in combination.

10. A modified DNA obtained by nucleotide modification of the DNA according to claim 8 or 9.

11. A DNA combination encoding the dsRNA combination of claim 4, which consists of the amino acid sequence of SEQ ID NO: 9-16.

12. The DNA combination of claim 11, wherein the DNA combination is SEQ ID NO:9 and SEQ ID NO:11 in the above-mentioned DNA combinations; the amino acid sequence of SEQ ID NO:10 and SEQ ID NO:11 in combination with the DNA shown in fig. 11; the amino acid sequence of SEQ ID NO:11 and SEQ ID NO:12 in combination with a DNA shown in seq id no; SEQ ID NO:11 and SEQ ID NO:16 in combination with a DNA shown in seq id no; SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12 and SEQ ID NO: 16; or SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO:16, or a combination of the DNAs shown in.

13. DNA encoding the siRNA of claim 6 or 7.

14. A delivery vector for inducing RNA interference comprising the dsRNA or combination of dsRNA, DNA or combination of DNA, or siRNA of any one of claims 1-13.

15. The delivery vector of claim 14, wherein the delivery vector is selected from the group consisting of viral vectors and non-viral vectors, preferably wherein the non-viral vector is selected from the group consisting of liposomes, plasmid vectors, phage vectors, preferably wherein the viral vector is selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, lentiviral vectors and hybrid viral vectors, more preferably wherein the viral vector is selected from the group consisting of adeno-associated viral vectors.

16. A kit for inducing RNA interference comprising the dsRNA or dsRNA combination, DNA or DNA combination, siRNA or delivery vehicle of any one of claims 1-15.

17. A method of obtaining a virus-free cell line from an organism or cell derived from a viral contamination comprising: obtaining cells from a virally contaminated organism or population of cells;

after washing the cells, introducing the dsRNA or combination of dsRNA, DNA or combination of DNA, siRNA of any one of claims 1-15, directly or via a delivery vector, into the obtained cells to induce RNA interference;

after washing and resuspending the cells, separating the cells into single cells or multiple cells;

the isolated single cell or plurality of cells are expanded in culture in a medium suitable for cell growth and division to produce single cell clones or multi-cell clones, thereby obtaining virus-free cell lines.

18. The method of claim 17, further comprising removing a portion of said cell clone or said culture medium to detect the presence of a virus.

19. The method of claim 17 or 18, wherein said virus comprises Sf-rhabdovirus.

20. The method according to any one of claims 17 to 19, wherein the cell line is derived from an insect, preferably a lepidopteran insect, more preferably spodoptera frugiperda.

21. The method of any one of claims 17-20, wherein the cell line is derived from a primary cell or cell line contaminated with a virus.

22. The method of claim 21, wherein the virus-contaminated cell line comprises Sf21 or Sf9 cells, and wherein the virus comprises Sf-rhabdovirus.

23. The method according to any one of claims 17-22, wherein said introducing comprises transfection or transduction, preferably transfection, more preferably lipofection.

24. The method of any one of claims 18-23, wherein the detecting comprises (a) RT-PCR; (b) RT-PCR and nested PCR; (c) quantitative RT-PCR; or (d) antibody-based detection techniques.

25. The method of any one of claims 17-24, wherein the method of isolating into a single cell or a plurality of cells comprises: limiting 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 using a micromanipulator, preferably limiting dilution cloning.

26. The method of claim 25, wherein the plurality of cells is 2-20 cells, preferably 3-10 cells, preferably 4-6 cells, more preferably 5 cells.

27. A virus-free cell line derived from a virus-contaminated organism or cell, the cell line obtained by the method of any one of claims 17-26.

28. The cell line of claim 27, wherein the cell line is derived from an insect contaminated with a virus.

29. The cell line of claim 28, wherein the cell line is derived from a lepidopteran insect.

30. The cell line of claim 29, wherein the lepidopteran insect comprises a spodoptera frugiperda, a cabbage looper, or a silkworm.

31. The cell line of claim 30, wherein the cell line is derived from a spodoptera frugiperda cell line.

32. The cell line of claim 31, which is deficient in Sf-rhabdovirus.

33. Use of a virus-free cell line as claimed in any one of claims 27 to 32 in a baculovirus-insect expression system.

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