Application of host factor MOV10 in development of rabies treatment medicine and vaccine production

Abstract

The invention discloses application of a host factor MOV10 in development of rabies treatment medicines and vaccine production, and belongs to the technical field of biological medicines. In order to research the influence of the host factor MOV10 on the replication of the RABV, the invention discovers that the overexpression host factor MOV10 coding gene can inhibit the replication of the RABV through the technologies of real-time fluorescent quantitative PCR (Polymerase Chain Reaction), Western Blotting, direct immunofluorescence and the like; then, a mov10 gene knockout cell line is successfully constructed through a CRISPR / Cas9 gene editing technology, the cell proliferation activity of the mov10 gene knockout cell line and the influence of the mov10 gene knockout cell line on RABV replication are researched, and it is found that stable knockout of the mov10 gene can remarkably promote replication of RABV CVS11 and SRV9. Technical support is provided for exploration of MOV10 biological functions and development and production of RABV inactivated vaccines and recombinant virus vaccines with RABV as carriers.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of host factor MOV10 in the development of rabies treatment drugs and vaccine production. Background Art

[0002] As an antiviral host factor, the innate immune restriction factor MOV10 has become a new research hotspot after SERINC5, APOBEC3 and Tetherin. With the deepening of research, MOV10 was found to be one of the proteins involved in the RNA-induced silencing complex (RISC), which can assist Argonaute 2 (AGO2) in binding to microRNA (miRNA) to regulate the storage and degradation of host protein mRNA and the genome of invading viruses, thereby maintaining the homeostasis of host cells and participating in the composition of the innate immune system. In recent years, the functions of MOV10 in regulating the life cycle of viruses have been reported one after another. MOV10 mainly regulates the life cycle of viruses through three pathways: interaction, P bodies and interferon. Interaction pathway: MOV10 can interact with the N-terminal domain of the Gag protein of human immunodeficiency virus type 1 (HIV-1) in an RNA-dependent manner and participate in the assembly process of the virus through this interaction; MOV10 can interact with the arm of the N protein of Severe Fever with Thrombocytopenia Syndrome Bunyavirus (SFTSV) in an RNA-independent manner, thereby inhibiting the assembly and replication of SFTSV. P body pathway: MOV10 binds to the NP protein of influenza A virus (IAV) and stores it in the cytoplasmic P body, inhibiting the nuclear import of the NP protein and the polymerase activity of vRNP, thereby inhibiting the replication of IAV. Interferon pathway: MOV10 induces the production of type I IFN through the RNA sensing pathway mediated by ikkε, thereby inhibiting the replication of type I herpesvirus. However, the research on the regulatory function of MOV10 on rabies virus (RABV) is still in a blank stage.

[0003] Rabies is a fatal zoonosis caused by RABV with severe neurological symptoms as the main feature. Rabies is currently prevalent worldwide, with approximately 59,000 deaths each year. It is one of the zoonoses that requires special attention. The prodromal symptoms of rabies are non-specific. Early local symptoms include paresthesia, itching, and pain at the site of viral invasion, which are believed to be the result of infection and inflammation of local sensory ganglia. In the later stages of disease infection, symptoms develop into brain dysfunction, abnormal behavior, ataxia, weakness, paralysis, breathing and swallowing difficulties, etc. The incubation period of rabies may range from a few weeks to a few months, and once rabies symptoms appear, the mortality rate of rabies is almost 100%. There is currently no specific drug for rabies, and the main treatment method after exposure is vaccination and immunoglobulin injection. Therefore, from the perspective of virus-host interaction, exploring the key host factors that regulate RABV replication can screen new therapeutic targets and rescue drugs for the clinical treatment of rabies.

[0004] At present, the rabies vaccine approved for use in my country is an inactivated vaccine, which has the advantage of high safety, but often requires multiple, high-dose immunizations to stimulate the body to produce effective protection. In addition, due to the simple genome structure of RABV, stable gene expression, short replication cycle, and a wide range of infection spectrum and other biological characteristics, it has become a preferred vector for recombinant viral vector vaccines. At present, recombinant vaccines for coronavirus, Nipah virus, Lassa fever virus, Zika virus, and peste des petits ruminants virus using RABV as a viral vector have been put into relevant research. Constructing a stable cell line that promotes RABV proliferation from the perspective of host factors can improve the production efficiency of RABV vaccines to a certain extent, reduce the cost of vaccine research and development, and provide a new tool for the development and production of RABV vaccines. Summary of the invention

[0005] In order to study the effect of natural immune restriction factor MOV10 on RABV replication, the present invention uses real-time fluorescence quantitative PCR, Western Blotting and direct immunofluorescence and other technical means to find that overexpression of the encoding gene of host factor MOV10 can inhibit the replication of RABV; then the mov10 gene knockout cell line is successfully constructed by CRISPR / Cas9 gene editing technology, and the cell proliferation activity of the mov10 gene knockout cell line and its effect on RABV replication are further studied. It is found that stable knockout of the mov10 gene can significantly promote the replication of RABV CVS11 and SRV9. The present invention provides a new target for the clinical treatment of rabies, and at the same time provides technical support for the development and production of RABV inactivated vaccines and recombinant viral vector vaccines with RABV as carriers.

[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention proposes the following technical solutions:

[0007] The first objective of the present invention is to provide an application of overexpressing the mouse mov10 gene in a host cell in inhibiting the replication of rabies virus, wherein the Gene ID of the mouse mov10 gene is 17454.

[0008] The second object of the present invention is to provide a preparation capable of promoting the expression of the mouse mov10 gene for use in the preparation of a drug for preventing and / or treating rabies virus infection, wherein the Gene ID of the mouse mov10 gene is 17454.

[0009] The third object of the present invention is to provide an application of inhibiting or down-regulating the expression of MOV10 protein in host cells in promoting the replication of rabies virus, wherein the amino acid sequence of the MOV10 protein has an accession number of NM_001163440.2 on NCBI.

[0010] The fourth object of the present invention is to provide a substance capable of inhibiting or down-regulating the expression of MOV10 protein in host cells for use in promoting the replication of rabies virus, wherein the amino acid sequence of the MOV10 protein has an accession number of NM_001163440.2 on NCBI.

[0011] In one embodiment of the present invention, the substance is a sgRNA targeting a gene encoding MOV10 protein or a recombinant vector comprising the sgRNA.

[0012] In one embodiment of the present invention, the sgRNA includes sgRNA-F having a nucleotide sequence as shown in SEQ ID NO.5 and sgRNA-R having a nucleotide sequence as shown in SEQ ID NO.6.

[0013] The fifth object of the present invention is to provide a cell line with a knockout of the mouse mov10 gene for use in improving the production efficacy of an inactivated RABV vaccine, wherein the Gene ID of the mouse mov10 gene is 17454.

[0014] The sixth object of the present invention is to provide a cell line with a knockout of the mouse mov10 gene for use in improving the production efficiency of a recombinant virus using rabies virus as a vector, wherein the Gene ID of the mouse mov10 gene is 17454.

[0015] In one embodiment of the present invention, the method for constructing the mouse mov10 gene knockout cell line is to transfect host cells with a recombinant vector containing sgRNA targeting the mouse mov10 gene.

[0016] In one embodiment of the present invention, the sgRNA includes sgRNA-F having a nucleotide sequence as shown in SEQ ID NO.5 and sgRNA-R having a nucleotide sequence as shown in SEQ ID NO.6.

[0017] In one embodiment of the present invention, the host cell is an N2A cell.

[0018] In one embodiment of the present invention, the rabies virus includes RABV CVS-11 and RABV SRV-9.

[0019] Beneficial effects of the present invention:

[0020] The present invention studies the effect of host protein MOV10 on RABV replication by real-time fluorescence quantitative PCR, Western Blotting, direct immunofluorescence and other methods, and confirms that MOV10 inhibits RABV replication as a negative regulatory factor. Subsequently, the mov10 gene knockout cell line was successfully constructed by the CRISPR / Cas9 gene editing system and it was confirmed that the mov10 gene knockout cell line has the same proliferation activity as the N2A cell line, and it was found that stable knockout of the mov10 gene can significantly promote the replication of RABV CVS11 and SRV9, and further found that the mov10 gene knockout cell line can improve the production efficiency of RABV inactivated vaccines and recombinant viruses with RABV as carriers, providing technical guarantee for the development and production of recombinant virus vector vaccines with RABV as carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic diagram of the recombinant plasmid pcDNA3.0-musMOV10;

[0022] Figure 2 TCID 50 Results of testing the effect of transient overexpression of mov10 gene on the titer of progeny virus of RABV CVS11 strain;

[0023] Figure 3 This is the result of RT-qPCR detection of the effect of transient overexpression of mov10 gene on the mRNA transcription level of N protein of RABV CVS11 strain; * indicates P < 0.05, *** indicates P < 0.001;

[0024] Figure 4 This is the result of Western Blotting to detect the effect of transient overexpression of mov10 gene on the replication of RABV CVS11 strain; Figure 4A in the figure is the result of Western Blotting to detect the effect of transient overexpression of mov10 gene on the level of N protein of RABV CVS11 strain. Figure 4 B is for Figure 4 The result of grayscale analysis of A in Figure 1 is shown in Figure 2; ** indicates P < 0.01, *** indicates P < 0.001;

[0025] Figure 5 Design diagram of sgRNA sequence targeting the third exon of mov10 gene;

[0026] Figure 6 This is the sequencing result diagram of the recombinant plasmid pMD18T-U6-sgRNA;

[0027] Figure 7 This is the effect diagram of co-transfection of pMD18T-U6-sgRNA and Cas9-eGFP; Figure 7 A in the figure is the empty cell control. Figure 7 B in the figure is the effect diagram of co-transfection of pMD18T-U6-sgRNA and Cas9-eGFP;

[0028] Figure 8 This is the result of Western Blotting to detect the expression of endogenous MOV10 protein in mov10 knockout cell lines;

[0029] Fig. 9 This is a comparison of the cell proliferation activity of wild-type N2A cell line and mov10 knockout cell line detected by CCK8; ns means no statistical difference;

[0030] Fig.10 This is the result of Western Blotting to detect the effect of stable knockout of mov10 gene on the expression level of RABV CVS11 N protein;

[0031] Fig.11 This is the result of RT-qPCR detection of the effect of stable knockout of mov10 gene on the transcription level of RABV CVS11 N mRNA; where ** indicates P < 0.01;

[0032] Fig.12 TCID 50 Results of detecting the effect of stable knockout of mov10 gene on the titer of RABV CVS11 progeny virus; ** indicates P < 0.01;

[0033] Fig.13 TCID 50 The results of testing the effect of replenishing MOV10 protein on the titer of RABV progeny viruses in mov10 knockout cell lines;

[0034] Fig.14 The results of RT-qPCR detection of the effect of MOV10 protein replenishment on the transcription level of RABV N protein mRNA in mov10 knockout cell lines; * indicates P < 0.05, *** indicates P < 0.001;

[0035] Fig.15 The figure is the result of Western Blotting to detect the effect of replenishing MOV10 protein on the replication of RABVCVS11 strain in mov10 knockout cell line; Fig.15 A in the figure is the result of Western Blotting to detect the effect of MOV10 protein replenishment on RABV N protein level in mov10 knockout cell lines. Fig.15 B is for Fig.15 The result of grayscale analysis of A in Figure 1 is shown in Figure 2; *** indicates P < 0.001;

[0036] Fig.16 This is the result of Western Blotting to detect the effect of stable knockout of mov10 gene on the expression level of RABV SRV9 N protein;

[0037] Fig.17 This is the result of RT-qPCR detection of the effect of stable knockout of mov10 gene on the transcription level of RABV SRV9 mRNAN; where ** indicates P < 0.01;

[0038] Fig.18 TCID 50 The results of detecting the effect of stable knockout of mov10 gene on the titer of RABV SRV9 progeny virus. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0040] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0041] The experimental methods involved in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, enzymes, cells, plasmids, etc. used are all commercially available unless otherwise specified.

[0042] The present invention performs statistical analysis on the results by t-test.

[0043] Example 1: Effect of transient overexpression of mov10 gene on replication of RABVCVS11 strain

[0044] 1. Construction of mov10 gene expression vector

[0045] The amino acid sequence of mouse MOV10 protein is registered at NCBI with the accession number NM_001163440.2. The full-length upstream primer musMOV10-F (with EcoRⅠ restriction site added) and the downstream primer musMOV10-R (with NotⅠ restriction site added) of mov10 were designed and synthesized according to the nucleotide sequence of mouse mov10 gene. The primer sequences are shown in Table 1. The target gene mov10 was amplified using cDNA extracted from N2A cells (mouse neuroma cells) as a template, and the target band was separated by 1% agarose gel electrophoresis, and the target fragment was recovered using a gel recovery kit (OMEGA, catalog number D2500-01). The pcDNA3.0 vector and the target gene fragment of mov10 were digested with Thermo rapid restriction enzymes EcoRⅠ and NotⅠ at 37℃ for 3h, and then the digested fragments were purified using an AxyPrep PCR cleaning kit (Axygen, catalog number AP-PCR-50). The pcDNA3.0 double-digested product was ligated with the mov10 target gene fragment using T4 DNA ligase (New England BioLabs, catalog number M0202L) according to the instructions, and DH5α competent cells were transformed. Single clones were selected for sequencing, and finally the target fragment was inserted into the pcDNA3.0 vector. The schematic diagram of the obtained recombinant plasmid pcDNA3.0-musMOV10 is shown in the figure. Figure 1 shown.

[0046] Table 1 Mov10 full-length primer sequence information

[0047]

[0048] 2. RABV CVS11 infection experiment after transient overexpression of mov10 gene

[0049] The N2A cell line was counted and expressed at 4.5 × 10 5 pcDNA3.0 and pcDNA3.0-musMOV10 were plated in 6-well plates and cultured in a 37°C incubator for 20 h. TMIn vitro DNA transfection reagents were used to transfect N2A cells (for transfection conditions and systems, refer to the instructions of the liposome). 24 hours after transfection, CVS11 was used to infect N2A cells transfected with pcDNA3.0 and N2A cells transfected with pcDNA3.0-musMOV10 recombinant plasmid at MOI = 0.1. After incubation at 37°C for 1 hour, the virus infection solution was discarded and fresh 5% fetal bovine serum DMEM medium was added. Cell supernatants and cell pellets were taken at 24h, 36h, and 48h after infection, and the cell pellets were divided into two parts according to a ratio of 3:7, which were used for subsequent RT-qPCR and Western Blotting detection, respectively, and stored at -80°C.

[0050] 3. TCID 50 Detection of the effect of transient overexpression of mov10 gene on the progeny virus titer of RABV CVS11 strain

[0051] The supernatant virus solution to be tested was diluted 9 times in a 10-fold gradient to 10 -9 At the same time, N2A cells that had grown to a monolayer were passaged and plated in 96-well plates at 100 μL / well. -9 ~10 -1 The diluted virus dilution solution was added to a 96-well plate at 50 μL / well in sequence, with 4 replicates for each dilution; the cells and virus solution were mixed evenly and cultured at 37°C for 48 hours; after 48 hours, the supernatant was discarded, and 150 μL / well of pre-cooled acetone with a volume fraction of 80% was added, and the plate was fixed at room temperature for 30 minutes; the fixative was discarded, 150 μL / well of PBST was added, the plate was washed, and this step was repeated 3 times, and the last time, the liquid in the well was gently tapped on paper to tap out; the diluted fluorescent antibody was added at 50 μL / well, and incubated at 37°C in the dark for 1 hour; the supernatant was discarded, 150 μL / well of PBST was added, the plate was washed, and this step was repeated 3 times, and the last time, the liquid in the well was gently tapped on paper to tap out; the 96-well plate was observed under a fluorescence microscope, the number of positive wells for each dilution was recorded, and the TCID 50 .

[0052] The results showed that compared with the group transfected with pcDNA3.0 plasmid, the virus titer of the RABVCVS11 strain progeny in the group transfected with pcDNA3.0-musMOV10 plasmid decreased, indicating that MOV10 protein overexpression can inhibit the proliferation of RABVCVS11 strain in N2A cells to a certain extent (see Figure 2 ).

[0053] 4. RT-qPCR detection of the effect of transient overexpression of mov10 gene on the transcription level of N protein mRNA of RABV CVS11 strain

[0054] Add 500 μL Trizol (Tian Gen, dp424) to each cell pellet, let it stand at room temperature for about 10 min to fully lyse the cells; then add 100 μL pre-cooled chloroform, mix thoroughly by inversion, let it stand on ice for 10 min, and centrifuge at 4°C 12000rpm for 15 min; transfer the transparent and clear uppermost liquid to a new 1.5mL EP tube, add an equal amount of pre-cooled isopropanol, mix thoroughly by inversion, let it stand on ice for 15 min, and centrifuge at 4°C 12000rpm for 15 min to reveal a white precipitate; discard the supernatant, add 1mL of 75% ethanol by volume (diluted with DEPC water), centrifuge at 4°C 12000rpm for 5 min, repeat twice, aspirate the ethanol remaining at the bottom of the tube, and dry on ice; add an appropriate amount of DEPC water to each EP tube, oscillate until the precipitate is completely dissolved, and measure the RNA concentration.

[0055] RNA was diluted to 200 ng / μL using DEPC water, and reverse transcribed using PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio, Catalog No. RR047A) to obtain cDNA (for reaction conditions and system, refer to the instruction manual of the kit).

[0056] Using cDNA as template, using PowerUp TM SYBR TM Green premix (applied biosystems, catalog number A25742) was used for qPCR (for reaction conditions and system, refer to the instructions of the kit). The RT-qPCR primer sequences of RABV N are shown in Table 2.

[0057] Table 2 RABV N RT-qPCR primer sequence information

[0058]

[0059] RT-qPCR results showed that compared with the control group transfected with pcDNA3.0 plasmid, the transcription level of RABV N protein mRNA in the group transfected with pcDNA3.0-musMOV10 plasmid was significantly reduced, indicating that MOV10 protein overexpression can significantly inhibit the replication of RABV CVS11 strain in N2A cells (see Figure 3 ).

[0060] 5. Western Blotting to detect the effect of transient overexpression of mov10 gene on the expression level of N protein of RABV CVS11 strain

[0061] 100 μL of cell lysis solution containing 1% PMSF was added to each cell pellet, shaken until there was no precipitation, and then placed on ice for 10 minutes; centrifuged at 4°C 12000rpm for 15 minutes, and the supernatant was collected as the total protein. N2A cells were used as controls, and SDS-PAGE electrophoresis was performed and the protein was transferred to the NC membrane, blocked with 5% skim milk powder at room temperature for 2 hours, and Anti-RABV N antibody (prepared and preserved in this laboratory) was used as the primary antibody, and intracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene (abclonal, AC002), incubated at 4°C overnight, and horseradish peroxidase-labeled goat anti-mouse IgG (bioworld, BS12478) was used as the secondary antibody for incubation at room temperature for 1 hour, and basic ECL luminescent liquid (Ya enzyme, SQ202L) was added for development in a dark room to detect the expression of RABV N protein. The results are shown in Figure 4 A in the figure. Grayscale analysis showed that the expression level of RABVN protein in the group transfected with pcDNA3.0-musMOV10 plasmid was significantly reduced compared with that in the group transfected with pcDNA3.0 plasmid, indicating that overexpression of MOV10 protein can significantly inhibit the replication of RABV CVS11 strain in N2A cells (see Figure 4 B).

[0062] Example 2: Construction and identification of mov10 knockout cell lines

[0063] 1. Design and synthesis of sgRNA

[0064] We used CRISPR-Cas9 technology to construct cell lines. We searched and downloaded the whole genome sequence information of mov10 from the NCBI website (GeneID: 17454), checked and designed sgRNA in the third exon region. We uploaded the sequence information of the third exon region to the online design website (CRISPOR (ucsc.edu)), selected the species mouse, designed the Cas9 protein sgRNA with the PAM region of "NGG" sequence, and selected the sgRNA with the highest positive sequence score and the lowest off-target rate (see Figure 5 In order to facilitate the connection between sgRNA and vector plasmid, the sticky ends of BbsⅠ restriction site were added on both sides of the primer (sequence information is shown in Table 3).

[0065] Table 3 MVO10-sgRNA primer sequence information

[0066]

[0067] 2. Construction and identification of recombinant plasmid pMD18T-U6-sgRNA

[0068] (1) Linearization of pMD18T-U6-sgRNA vector

[0069] First, the pMD18T-U6-huANPsgRNA-1 (see the invention name of the application of ANP32 protein in maintaining influenza virus polymerase activity in the host, the application number is CN201810177710.X) recombinant plasmid retained in the laboratory was digested and recovered with the restriction endonuclease BbsⅠ. The digestion system is: pMD18T-U6-huANPsgRNA-1 2.5μg, BbsⅠ (thermo, ER1011) 2.5μL, 10× digestion buffer 5μL, double distilled water to make up 50μL. The reaction system was shaken, mixed and centrifuged, and then placed in a 37℃ water bath for 3h, and the digestion product was recovered by a DNA purification kit (uelandy, UE-PCR-250).

[0070] (2) sgRNA annealing

[0071] At the same time, sgRNA was annealed. The annealing system was: sgRNA-F 5μL, sgRNA-R 5μL, 10×NEBbuffer 3 (NEB, B7203S-5mL) 5μL, double distilled water to 50μL. The reaction system was shaken and centrifuged, then placed in a PCR instrument for annealing reaction. The reaction system was: 95℃5min; 25℃30min.

[0072] (3) Construction and identification of recombinant plasmid pMD18T-U6-sgRNA

[0073] Connect the linearized pMD18T-U6 vector to the annealed sgRNA product. The connection system is: 2×solutionⅠ (TAKARA, 6002) 5μL, pMD18T-U6 linearized vector 0.5μL, annealed sgRNA product 4.5μL. After the reaction system is shaken and centrifuged, connect overnight at 4°C. Take 5μL of the connection product and transform it into 50μL DH5α competent medium (TAKARA, 9057), evenly spread it on an ampicillin-resistant agar plate, and invert and culture it at 37°C overnight. Pick a single clone positive plasmid for sequencing, and follow the instructions of the endotoxin-free mini-extraction kit (Kangwei Century, CW2106) to sequence the correctly sequenced recombinant plasmid (see Figure 6 ) for endotoxin-free extraction.

[0074] 3. Construction and identification of mov10 knockout cell lines

[0075] (1) Cell transfection

[0076] Well-cultured N2A cells were cultured at 4.5×10 5 The density of cells / mL was evenly plated in 6-well plates. When the cells grew to 60%-70%, the polyjet TMTransfection reagent instructions: co-transfect the recombinant plasmid pMD18T-U6-sgRNA and the eukaryotic plasmid pMJ920 (Addgene plasmid #42234) expressing Cas9-GFP protein into N2A cells at a transfection ratio of 2:1 (1.4 μg: 0.7 μg) (see Figure 7 ), 37°C, 5% CO2. After 5 hours of transfection, fresh DMEM medium with 5% fetal bovine serum was replaced and culture continued.

[0077] (2) Screening of mov10 knockout cell lines

[0078] The cells cultured for 36 h were digested normally using 0.25% trypsin (CORNING, 25-053-CI), and 10% fetal bovine serum cell culture medium was added to terminate the digestion. The digested cells were blown and mixed, and the cell suspension was collected and filtered using a 70-mesh copper screen (BIOFIL, CSS013070), and the filtrate was collected in a sterile 1.5 mL centrifuge tube (axygen, MCT-150C).

[0079] Cells with GFP-labeled fluorescence were screened by flow cytometry and placed into 96-well plates according to the principle of 1 cell per well. After 10 days of sorting, monoclonal cells (single cell clusters) were found under a microscope and marked. After the cells were fully grown, the monoclonal cell clusters were transferred to 24-well plates, 12-well plates, and 6-well plates in turn. Finally, the cells were collected for knockout verification.

[0080] (3) Western blotting to detect the expression of MOV10 in mov10 knockout cell lines

[0081] The expression of MOV10 protein was detected by Western blotting using rabbit anti-MOV10 (abclonal, A7227). The specific steps are shown in step 5 of Example 1. The results showed that the endogenous MOV10 protein was basically not expressed in the mov10 knockout cell line, indicating that the mov10 knockout cell line was successfully constructed (see Figure 8 ).

[0082] 4. Cell activity detection of mov10 knockout cell lines

[0083] The wild-type N2A cell line and the selected mov10 knockout cell line were respectively cultured at 4.5×10 5The cells were plated evenly at a density of 100 μL / mL in a 96-well plate. Each cell type was plated in triplicate, with 200 μL cells per well, and cultured at 37°C and 5% CO2. The culture medium was aspirated and discarded 24h, 36h, and 48h after plating, and 100 μL of a mixed solution of CCK8 (APEBIO, K1018) and high-glucose culture medium (CCK8: DMEM = 1: 9) was added to each well, and cultured at 37°C and 5% CO2 for 1h. The absorbance was measured at 450nm as the detection wavelength and 630nm as the reference wavelength to detect the effect of mov10 knockout on cell proliferation activity.

[0084] The results showed that there was no significant difference in cell proliferation activity between the mov10 knockout cell line and the wild-type N2A cell line (see Fig. 9 ).

[0085] Example 3: Effect of mov10 knockout cell lines on RABV replication

[0086] 1. Effect of stable knockout of mov10 gene on the replication of RABV CVS11 strain

[0087] (1) RABV CVS11 infection experiment after stable knockout of mov10 gene

[0088] The wild-type N2A cell line and the mov10 knockout cell line were counted and expressed as 6 × 10 5 100 μg / mL were plated in 6-well plates. After culturing in a 37°C incubator for 20 h, RABV CVS11 was used to infect wild-type N2A cell lines and mov10 knockout cell lines at an MOI of 0.1. After 1 h at 37°C, the virus infection solution was discarded and fresh 5% fetal bovine serum medium was added. At 36 h after infection, the cell supernatant and cell pellet were taken, and the cell pellet was divided into two parts at a ratio of 3:7 for subsequent RT-qPCR and Western Blotting detection, respectively, and stored at -80°C.

[0089] (2) Western Blotting to detect the effect of stable knockout of mov10 gene on the expression level of N protein of RABV CVS11 strain

[0090] Western Blotting was used to detect the expression level of RABV N protein. The specific steps are shown in step 5 of Example 1. The results showed that compared with the wild-type N2A cell group, the expression level of RABV N protein in the mov10 knockout cell group was significantly increased, indicating that stable knockout of the mov10 gene can significantly promote the replication of RABV CVS11 (see Fig.10 ).

[0091] (3) RT-qPCR detection of the effect of stable knockout of mov10 gene on the transcription level of RABV N protein mRNA

[0092] RT-qPCR was used to detect the transcription level of RABV N protein mRNA. The specific steps are shown in step 4 of Example 1. The results showed that compared with the wild-type N2A cell group, the transcription level of RABV N protein mRNA in the mov10 knockout cell group was significantly increased, indicating that stable knockout of the mov10 gene can significantly promote the replication of RABV CVS11 ( Fig.11 ).

[0093] (4)TCID 50 Detection of the effect of stable knockout of mov10 gene on RABV progeny virus titer

[0094] Using TCID 50 The RABV virus titer was detected, and the specific steps are shown in step 3 of Example 1. The results showed that compared with the wild-type N2A cell group, the RABV CVS11 progeny virus titer in the mov10 knockout cell group was significantly increased, indicating that stable knockout of the mov10 gene can significantly promote the proliferation of RABV CVS11 (see Fig.12 ).

[0095] 2. Effect of complementation of MOV10 protein in mov10 knockout cell lines on the replication of RABV CVS11

[0096] (1) RABV CVS11 infection experiment after MOV10 protein supplementation

[0097] The wild-type N2A cell line and the mov10 knockout cell line were counted and expressed as 4.5 × 10 5 pcDNA3.0 was plated in 6-well plates and cultured in a 37°C incubator for 20 h. TM In vitro DNA transfection reagents were used to transfect wild-type N2A cell lines and mov10 knockout cell lines. 1 μg of pcDNA3.0-musMOV10 was transfected into the cell lines through polyjet TM Transfect the mov10 knockout cell line with an in vitro DNA transfection reagent (for transfection conditions and systems, refer to the instructions of the liposome). 24 hours after transfection, CVS11 was used to infect the wild-type N2A cell line transfected with pcDNA3.0, the mov10 knockout cell line transfected with pcDNA3.0, and the mov10 knockout cell line transfected with pcDNA3.0-musMOV10 at an MOI of 0.1. After incubation at 37°C for 1 hour, the virus infection solution was discarded and fresh 5% fetal bovine serum DMEM culture medium was added. The cell supernatant and cell pellet were taken 36 hours after infection and stored at -80°C.

[0098] (2)TCID 50Detection of the effect of complementation of MOV10 protein on the progeny virus titer of RABV CVS11 strain

[0099] Using TCID 50 The RABV virus titer was detected, and the specific steps are shown in step 3 of Example 1. The results showed that the RABV progeny virus titer showed a downward trend after MOV10 was replenished in the mov10 knockout cell line, indicating that replenishing MOV10 protein can eliminate the promoting effect of RABV proliferation in the mov10 knockout cell line (see Fig.13 ).

[0100] (3) RT-qPCR detection of the effect of complementation of MOV10 protein on the transcription level of N protein mRNA of RABV CVS11 strain

[0101] The transcription level of RABV N protein mRNA was detected by RT-qPCR, and the specific steps are shown in step 4 of Example 1. The results showed that the transcription level of RABV N protein mRNA was significantly reduced after MOV10 was replenished in the mov10 knockout cell line, indicating that replenishing MOV10 protein can restore the inhibitory effect on RABV replication (see Fig.14 ).

[0102] (4) Western Blotting to detect the effect of complementation of MOV10 protein on the expression level of N protein of RABV CVS11 strain

[0103] Western Blotting was used to detect the expression level of RABV N protein. The specific steps are shown in step 5 of Example 1. The results are shown in Fig.15 A in Figure 2. Grayscale analysis showed that the expression level of RABV N protein was significantly reduced after MOV10 was replenished in the mov10 knockout cell line, indicating that replenishing MOV10 protein can relieve the promotion of RABV replication in the mov10 knockout cell line (see Fig.15 B).

[0104] 3. Effect of stable knockout of mov10 gene on the replication of RABV SRV9 strain

[0105] (1) RABV SRV9 infection experiment after stable knockout of mov10 gene

[0106] The wild-type N2A cell line and the mov10 knockout cell line were counted and expressed as 6 × 10 5100 μg / mL was plated in a 6-well plate. After 20 h of incubation at 37°C, wild-type N2A cells and mov10 knockout cells were infected with RABV SRV9 at MOI = 0.1. After 1 h of incubation at 37°C, the virus infection solution was discarded and fresh 5% fetal bovine serum DMEM medium was added. At 36 h after infection, the cell supernatant and cell pellet were taken, and the cell pellet was divided into two parts according to 3:7, which were used for subsequent RT-qPCR and Western Blotting detection, respectively, and stored at -80°C.

[0107] (2) Western Blotting to detect the effect of stable knockout of mov10 gene on the expression level of RABV N protein

[0108] The expression level of RABV N protein was detected by Western Blotting. The specific steps are shown in step 5 of Example 1. The results showed that compared with the wild-type N2A cell group, the expression level of RABV N protein in the mov10 knockout cell group was significantly increased, indicating that stable knockout of the mov10 gene can significantly promote the replication of RABV SRV9 (see Fig.16 ).

[0109] (3) RT-qPCR detection of the effect of stable knockout of mov10 gene on the transcription level of RABV N protein mRNA

[0110] The transcription level of RABV N protein mRNA was detected by RT-qPCR, and the specific steps are shown in step 4 of Example 1. The results showed that compared with the wild-type N2A cell group, the transcription level of RABV N protein mRNA in the mov10 knockout cell group was significantly increased, indicating that stable knockout of the mov10 gene can significantly promote the replication of RABV SRV9 (see Fig.17 ).

[0111] (4)TCID 50 Detection of the effect of stable knockout of mov10 gene on RABV progeny virus titer

[0112] Using TCID 50 The RABV virus titer was detected, and the specific steps are shown in step 3 of Example 1. The results showed that compared with the wild-type N2A cell group, the RABV SRV9 progeny virus titer in the mov10 knockout cell group showed an upward trend, indicating that stable knockout of the mov10 gene can promote the proliferation of the RABV SRV9 strain to a certain extent (see Fig.18 ).

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of overexpression of mouse mov10 gene in host cells in inhibiting rabies virus replication, characterized in that: The Gene ID of the mouse mov10 gene is 17454.

2. Use of a preparation capable of promoting the expression of mouse mov10 gene in the preparation of a drug for preventing and / or treating rabies virus infection, characterized in that: The Gene ID of the mouse mov10 gene is 17454.

3. Application of inhibiting or down-regulating the expression of MOV10 protein in host cells in promoting rabies virus replication, characterized in that: The accession number of the amino acid sequence of the MOV10 protein in NCBI is NM_001163440.

2.

4. Use of a substance capable of inhibiting or down-regulating the expression of MOV10 protein in a host cell in promoting the replication of rabies virus, characterized in that: The accession number of the amino acid sequence of the MOV10 protein in NCBI is NM_001163440.

2.

5. The use according to claim 4, characterized in that: The substance is a sgRNA targeting a MOV10 protein encoding gene or a recombinant vector containing the sgRNA.

6. The use according to claim 5, characterized in that: The sgRNA includes sgRNA-F whose nucleotide sequence is shown in SEQ ID NO.5 and sgRNA-R whose nucleotide sequence is shown in SEQ ID NO.

6.

7. Application of a mouse mov10 gene knockout cell line in improving the production efficacy of an inactivated rabies virus vaccine, characterized in that: The Gene ID of the mouse mov10 gene is 17454.

8. Use of a mouse mov10 gene knockout cell line in improving the production efficiency of a recombinant virus using rabies virus as a vector, characterized in that: The Gene ID of the mouse mov10 gene is 17454.

9. The use according to any one of claims 7 or 8, characterized in that: The method for constructing the mouse mov10 gene knockout cell line is to transfect host cells with a recombinant vector containing sgRNA targeting the mouse mov10 gene.

10. The use according to claim 9, characterized in that: The host cell is N2A cell.

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