SOD2 knockout cell line and application thereof in anti-poxvirus

Abstract

The invention discloses an SOD2 knockout cell line and application of the SOD2 knockout cell line in anti-poxvirus, and relates to the technical field of antiviral research. According to the invention, sgRNA of targeted superoxide dismutase 2 (SOD2) is designed, the sequence of the sgRNA is shown as SEQ ID NO.1-SEQ ID NO.2, then a stable SOD2 knockout human-derived non-small cell lung cancer A549 cell line is established by combining CRISPR / Cas9 gene editing with a lentiviral vector delivery technology, single-cell cloning is obtained through multiple rounds of puromycin screening, and the SOD2 gene knockout A549 cell strain is successfully constructed. Vaccinia virus Tian Tan strain infection shows that the number of plaques of SOD2 knockout cells is obviously greater than that of the plaques (about 2.3 times) of normal cell infection, and the plaques are relatively large, so that fine SOD2 has the effect of limiting intercellular transmission of poxvirus. The invention lays a foundation for research and preparation of antiviral drugs.

Description

Technical Field

[0001] This invention belongs to the field of antiviral research technology, specifically relating to an SOD2 knockout cell line and its application in anti-poxvirus. Background Technology

[0002] Viral infection is a complex biological process in which the interaction between the virus and the host not only mediates viral replication and spread but also triggers the activation of intracellular signaling pathways. Existing evidence suggests that viral pathogens can induce oxidative stress, leading to the abnormal accumulation of reactive oxygen species (ROS) within cells. ROS are primarily produced as byproducts of aerobic metabolism and can directly damage host cellular components. This oxidative damage can trigger cellular dysfunction and apoptosis, while simultaneously regulating redox-sensitive signaling pathways and the production of inflammatory mediators. Previous studies in this invention have shown that in vitro EBV infection can induce oxidative stress in B lymphocytes and epithelial cells, possibly through upregulation of Nox2 followed by ROS production mediated by NADPH oxidase in B lymphocytes. Studies have confirmed that influenza virus is a potent inducer of oxidative stress. Specifically, influenza A virus (IAV) infection disrupts glutathione redox balance and increases ROS levels in lung epithelial cells. NADPH oxidases, particularly the NOX4 subtype, play a major role in IAV-induced ROS production. These ROS activate the p38 and ERK1-2 MAPK pathways, thereby promoting nuclear export of the viral ribonucleoprotein (vRNP) complex to regulate the viral life cycle. GO and MetaCore pathway analyses of MPXV-infected human cells revealed affected pathways and genes, including Notch signaling, CD40, hypoxia-inducible factor-1α (HIF-1α), IL-1, IL-6, non-apoptotic Fas receptor (CD95), phosphatidylinositol 3-kinase (PI3K), nuclear factor-κB (NF-κB), the MAPK cascade, and oxidative stress-related signaling pathways. These alterations indicate that MPXV triggers a strong host cellular response. Crucially, virus-induced oxidative stress significantly modulates host immune regulation. Given that this unique pathogenic mechanism involves oxidative sensitivity signaling pathways, antioxidant interventions represent promising antiviral targets.

[0003] To maintain intracellular reactive oxygen species (ROS) homeostasis, eukaryotic cells utilize a complex antioxidant defense system. A core mechanism is the superoxide dismutase (SOD) family, particularly SOD2 located in mitochondria in aerobic cells, which maintains redox homeostasis by scavenging superoxide radicals (O2-). Notably, viral infection modulates SOD2 activity through multiple mechanisms—either inhibiting or disrupting its antioxidant function—thereby undermining host antiviral defenses and promoting viral pathogenicity. Although the importance of SOD2 in antioxidant defense is well-established, its mechanistic role in viral infection and innate immunity remains incompletely understood. Therefore, elucidating the dual function of SOD2 in viral infection is expected to deepen our understanding of virus-host interactions and provide a basis for novel therapeutic strategies targeting the regulation of oxidative homeostasis. Summary of the Invention

[0004] To address the aforementioned technical challenges, this invention utilizes VCV as a model virus and employs CRISPR-Cas9 gene editing technology combined with a lentiviral vector system to successfully construct an SOD2 gene-knockout human lung adenocarcinoma A549 cell line. The aim is to explore the function of SOD2 in the complex interactions between poxviruses and host cells. Experiments revealed that SOD2 gene knockout significantly increased viral plaque formation after VCV infection, indicating that SOD2 gene knockout promotes viral transmission and replication between cells. This is the first time that the crucial role of SOD2 in limiting VCV intercellular transmission has been revealed, providing new experimental evidence for understanding the function of SOD2 in antiviral immunity.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] One objective of this invention is to provide an sgRNA, the sequence of which is shown in SEQ ID NO.1~SEQ ID NO.2.

[0007] A second objective of this invention is to provide an expression vector containing the sgRNA.

[0008] The third objective of this invention is to provide a CRISPR / Cas9 system, which includes sgRNA and Cas9 protein.

[0009] The fourth objective of this invention is to provide an SOD2 gene knockout method for non-disease treatment purposes, which includes the step of using the sgRNA described above.

[0010] Furthermore, the SOD2 gene knockout method includes the following steps:

[0011] (1) Primer design: The primer sequences are shown in SEQ ID NO.1~SEQ ID NO.2;

[0012] (2) Plasmid construction: This includes the step of digesting the vector with the restriction endonuclease BsmBI;

[0013] (3) Lentiviral packaging and stable strain screening: The plasmid obtained in step (2) was transfected into 293T cells to obtain lentiviral supernatant, which was then used to infect A549 cells. Finally, the cells were screened using culture medium containing puromycin.

[0014] (4) Cell enzyme digestion and sequencing identification.

[0015] Furthermore, the enzyme digestion vector includes LentiCRISPRv2.

[0016] The fifth objective of this invention is to provide the application of the sgRNA, the expression vector, the CRISPR / Cas9 system, and / or any of the SOD2 gene knockout methods in the targeted knockout of the SOD2 gene.

[0017] The sixth objective of this invention is to provide an SOD2 knockout cell line, which is prepared by using the sgRNA, the expression vector, the CRISPR / Cas9 system and / or any of the SOD2 gene knockout methods.

[0018] The seventh objective of this invention is to provide the application of the sgRNA, the expression vector, the CRISPR / Cas9 system, any of the SOD2 gene knockout methods, and / or the cell lines in SOD2 antiviral research and / or antiviral drug preparation.

[0019] Furthermore, the virus includes vaccinia virus.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention designs a specific sgRNA targeting exon 2 of the SOD2 gene (NM_000636.4). Using CRISPR-Cas9-mediated gene editing technology combined with a lentiviral delivery system, an SOD2 gene knockout cell line was successfully established. Complete gene deletion was confirmed by DNA sequencing and Western blotting, validating the knockout at both the genomic and protein levels. The SOD2 knockout cell model provides a reliable platform for functional studies. This invention provides a fundamental framework for exploring the role of SOD2 in antiviral activity, and the established SOD2 knockout model provides a crucial experimental platform for elucidating SOD2-mediated antiviral defense mechanisms. Furthermore, elucidating the interaction between poxvirus and the host will accelerate the development of targeted antiviral strategies and enhance preparedness against novel threats from poxviruses. Attached Figure Description

[0022] Figure 1 This document describes the construction process of the SOD2 knockout cell line in Example 1 of the present invention, including: (A) the location of SOD2 on the chromosome and the sequence information of the sgRNA insertion region; (B) the cloning map information of the pLenti-sgRNA plasmid expressing sgRNA, Cas9, and puromycin resistance; and the sequencing results of the lentiCRISPR-SOD2-sgRNA recombinant plasmid; (C) the identification of T7 endonuclease I in A549-SOD2 KO cells; (D) the identification of SOD2 expression in A549-SOD2 KO cells; and (E) the sequencing of A549-SOD2 KO cells.

[0023] Figure 2 The verification process for SOD2 knockout promoting VCV infection in Example 1 of this invention includes: (A) 24 hours after infection, cells were fixed with 4% paraformaldehyde (PFA) and stained with crystal violet. (B) The number of viral plaques... P<0.05. (C) Western blot results showed that SOD2 knockout promoted VCV protein expression. (D) Quantitative analysis of VCV protein levels was performed by Western blotting. β-actin was used as an internal control for standardization. Data are expressed as mean ± standard error (n = 3 independent experiments). P<0.01. (E) A549-SOD2 knockout cells and control cells were infected with GFP-expressing VCV at different multiplicity of infection. One hour after infection, the cells were washed with PBS and cultured in medium containing 0.5% methylcellulose. Photographs were taken 24 hours later. (F) Size of viral plaques. P < 0.0001. (G) Cells stained with crystal violet 24 hours after infection. (H) Number of viral plaques as shown in Figure G. P<0.05; P < 0.01.

[0024] Figure 3 The in vivo antiviral experiment results of SOD2 in Example 1 of this invention are as follows: (A) In vivo antiviral procedure. A liposome-plasmid complex (30 μg) was subcutaneously injected into the back of a rabbit. Two days later, vaccinia virus (VACV, 1×10⁻⁶) was intradermally injected at the same injection site. 6 (PFU, 100 μL). (B) Formation of pox spots on the back of rabbits. Formation and development of pox spots on the back of rabbits in the SOD2 treatment group and the control group at different time points after challenge. Detailed Implementation

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0026] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0027] Example 1

[0028] 1. Materials and Methods

[0029] 1.1 Cells and Viruses

[0030] HEK293T cells were cultured in DMEM (DMEM; Gibco, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin. A549 cells were cultured in DMEN / F12 nutrient mixture (DMEM / F12; Gibco, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin. All cells were cultured at 37°C in a 5% CO2 incubator and were provided by the Stem Cell Bank of the Chinese Academy of Sciences. Vaccination virus (VACV) was preserved in our laboratory.

[0031] 1.2 Reagents and Antibodies

[0032] GoldBand Plus trichrome prestained protein molecular weight standards (8-180 kDa) (YEASEN, #20350ES72, China); SOD2 polyclonal antibody (Proteintech, #24127-1-AP, China); β-actin polyclonal antibody (Proteintech, #20536-1-AP, China); GFP (4B10) mouse monoclonal antibody (CST, #2955, USA).

[0033] 1.3 Primer Design

[0034] sgRNA: The coding region (CDS) sequence of the SOD2 gene (gene ID: 6648) was retrieved online through the NCBI website to determine the exon region distribution. sgRNA sequences targeting the exon regions of the SOD2 gene were designed and obtained using an online website (https: / / portals.broadinstitute.org / gpp / public / ) (Table 1). The sgRNA sequences were synthesized by the manufacturer.

[0035] Table 1. SOD2 sgRNA sequence design

[0036]

[0037] 1.4 Plasmid Construction

[0038] 1 μL each of forward and reverse primers (10 μmol / L), 1 μL of 10×T4 ligase buffer (containing ATP), 0.5 μL of T4 polynucleotide kinase (T4 PNK), and nuclease-free water were added to a total volume of 10 μL. The reaction mixture was incubated at 37 °C for 30 min, 95 °C for 5 min, and then cooled to 25 °C in a gradient of 5 °C / min. The annealed product was diluted 200-fold before use. The LentiCRISPRv2 vector was digested with the restriction endonuclease BsmB I and reacted at 37 °C for 30 min, followed by purification using agarose gel electrophoresis. 50 ng of linearized product, 1 μL of sgRNA annealing product, 1 μL of 2×Quick Ligase Buffer, and nuclease-free water were added to a total volume of 10 μL. Finally, 1 μL of Quick Ligase was added, and the mixture was incubated at room temperature for 10 min. The ligation product was transferred to Stble competent cells and spread evenly on solid medium containing Amp+. After 12 h, single colonies were picked for PCR identification. Plasmids of correctly identified strains were extracted using an endotoxin-free plasmid mini-prep kit to obtain plasmid lentiCRISPR-SOD2-sgRNA. The plasmid was then sent to Qingke Company for sequencing to obtain a recombinant plasmid expressing sgRNA with the correct sequence.

[0039] 1.5 Lentiviral Packaging and Screening of Stable Transformed Strains

[0040] The density of 293T cells was adjusted to approximately 2 × 10⁻⁶ cells using DMEM medium containing 10% fetal bovine serum. 5 10 cells / mL, inoculated into 6-well plates, 37°C o C. Incubate overnight in a 5% CO2 incubator. When the cell density reaches 70%-80%, co-transfect pLenti-SOD2-sgRNA and pLenti-control-sgRNA with pMD2.G and pCMV-dR8.2 dvpr plasmids, respectively, into 293T cells. Incubate for 6 h, then replace with fresh complete culture medium and continue culturing for 48 h. Collect the cell supernatant, filter through a 0.45 µM filter; this supernatant contains lentivirus. Infect A549 cells with 2 mL of lentiviral supernatant containing polybrene. 37 o Incubate at C for 3-6 h, then replenish with complete culture medium. Continue infection for 48 h, then aspirate the culture medium containing lentivirus and replace with fresh culture medium containing 2 μg / mL puromycin for screening.

[0041] 1.6 Cell Enzyme Digestion and Sequencing Identification

[0042] Genomic DNA was extracted from single-clonal cell lines, and the target gene fragment was amplified by PCR. PCR reaction conditions: 94 ℃ for 2 min; 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 60 s, for a total of 35 cycles; 72 ℃ for 5 min. 1 μL of T7EI was added to the annealed system, and the mixture was incubated at 37ºC for 30 min. The digestion reaction was terminated by heating at 85ºC for 15 min. The digestion products were subjected to agarose gel electrophoresis, photographed, and the digestion effect was analyzed. The PCR products were recovered, ligated into pMD 19-T Vector, transformed into DH5α competent cells, plated, amplified, and then plasmids were extracted and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0043] 1.7 SOD2 protein expression in A549 cells after SOD2 gene knockout

[0044] Control A549 cells and knockout A549 cells were collected and lysed using Western spectroscopy and IP lysis buffer. Protein quantification was performed using the BCA method. The protein loading volume was calculated based on the protein concentration. An appropriate amount of 5×SDS loading buffer was added, and the cells were heated at 100°C for 10 min to denature the proteins. After brief centrifugation, the supernatant was collected for SDS-PAGE. After electrophoresis, the supernatant was transferred to an NC membrane using a wet transfer method and blocked with 5% skim milk powder for 1 h. The membrane was then incubated with primary antibody at room temperature for 2 h, washed three times with 1×TBST for 10 min each time, and incubated with secondary antibody horseradish peroxidase-labeled goat anti-rabbit IgG for 30 min at room temperature, washed three times with 1×TBST for 10 min each time. The membrane was developed using ECL chemiluminescence and photographed using a gel imaging system. Grayscale analysis was performed using ImageJ software to calculate the relative protein content, validating the protein expression level of the SOD2 gene knockout A549 cell line.

[0045] 1.8 Plaque Emission Experiment

[0046] A549-SOD2 KO and control cells were mixed at a ratio of 2×10⁻⁶. 5Cells were seeded at a density of 1 / mL in 12-well plates. After cell adhesion, the virus solution was added to the culture plate and incubated at 37°C for 2 h. The plate was then replaced with 2% DMEM / F12 and cultured for another 24 h, with replicates. Culture was terminated once obvious plaques appeared. The supernatant was collected to detect the viral titer, and some wells were collected for protein extraction to detect viral expression at the protein level. The remaining wells were fixed with 4% paraformaldehyde for 15 min, washed twice with PBS, and stained with 300 μL of crystal violet staining solution at room temperature for 30 min. After thorough washing with distilled or tap water, the cells were ready for observation and photography. A549-SOD2 KO and control cells were infected with GFP-expressing VCV at different infection multiples. After 24 h, the cells were observed and photographed under a fluorescence microscope. Plaque formation was recorded after crystal violet staining.

[0047] 1.9 Animal Experiments

[0048] New Zealand white rabbits (weighing 2.5-3.0 kg, purchased from Guangdong Provincial Medical Laboratory Animal Center) underwent dorsal hair removal, followed by intradermal injection of liposome-plasmid complex (30 μg). Two days later, poxvirus (1×10⁻⁶) was inoculated intradermally at the same injection site. 6 PFU (100 μL). Subsequently, the formation and development of pox spots on the rabbit's back were continuously observed at different time points after infection.

[0049] 1.10 Statistical Analysis

[0050] Data are expressed as mean ± standard error (SEM) and are from 3 or 6 replicates. Data represent the results of three independent experiments. Statistical analysis was performed using GraphPad Prism 10.4.1 software. Statistical comparisons were performed using one-way ANOVA combined with Dunnett's multiple comparison test. ns indicates no significant difference; P < 0.05; P < 0.01; P < 0.001; P < 0.0001.

[0051] 2 Experimental Results

[0052] 2.1 Construction and identification of knockout cells

[0053] The SOD2 gene is located on chromosome 6 at position 6q25.3 and has 5 exons. This invention selects the region of the second exon of SOD2 ( Figure 1A) Using an online tool (https: / / portals.broadinstitute.org / gpp / public / ), an sgRNA sequence was designed. Then, based on the lentiCRISPR v2 vector information and the selected sgRNA gene sequence, specific primers were designed. This invention observed two adjacent BbsI restriction enzyme sites between the 5' end of the gRNA scaffold sequence and the U6 promoter. After enzyme digestion, two sticky ends are formed, with an additional GTGG 3' end on the left and a sticky GTGG end on the right. In synthesizing the sgRNA sequence, this invention allows direct insertion of the target sequence into the vector by simply adding a CACC sticky sequence to the left 5' end and the right 3' end. Sequencing of the ligation product showed that the sgRNA sequence was consistent with the expected sequence. Figure 1 B).

[0054] The constructed plasmid plenti-SOD2-sgRNA, along with the helper plasmids pCMV-dR8.2 dvpr and pMD2.G, was co-transfected into 293T cells for lentiviral packaging. Lentivirally infected cells were selected with puromycin and validated by restriction endonuclease digestion and sequencing. Genomic DNA was then extracted from these cells. Primers flanking the sgRNA target site were designed for PCR amplification. The resulting amplified fragments were digested with T7 endonuclease I (T7EI). T7EI recognizes and cleaves heteroduplex DNA formed by hybridization of wild-type and mutant strands (containing mismatched sequences, in this case, a 5-base loop). Homozygous double-stranded DNA (containing only wild-type or only mutant strands) remains intact and is not cleaved. Figure 1 C). After successfully knocking out the SOD2 gene, A549 cells were expanded and cultured, and proteins were collected for Western blot analysis to detect SOD2 protein expression. Compared with control cells, SOD2 protein expression was completely absent in A549-SOD2 KO cells. Figure 1 D). Subsequently, the genomic region targeted by sgRNA was amplified by PCR and followed by routine Sanger sequencing. The amplified fragment was approximately 400 bp in size. Sequencing analysis revealed a T-base insertion in exon 2 of the SOD2 gene in the knockout cells compared to the wild-type sequence. This insertion resulted in a frameshift mutation, producing a premature stop codon, which leads to premature termination of translation. Figure 1 E). The above results indicate that the SOD2 gene knockout cell line has been successfully constructed.

[0055] 2.2 Knockdown of SOD2 promotes VCV infection

[0056] Viral transmission within a host system primarily occurs through two distinct mechanisms: extracellular particle diffusion and direct intercellular transmission. The latter relies on tight junctions or viral synapses, significantly enhancing the efficiency of viral transfer between adjacent cells. Intercellular transmission allows viruses to bypass the rate-limiting steps of liquid-phase diffusion, directly infecting target cells from adjacent infected cells. Vaccine viruses utilize actin-driven motility to enhance this transmission mode, forming characteristic viral plaques through a cell-contact-dependent mechanism.

[0057] To assess the effect of SOD2 gene knockout on VACV infection, A549-SOD2 KO cells and control cells were seeded with VACV at a low multiplicity of infection (MOI = 0.1 PFU / cell). 24 h after infection, cells were fixed, stained with 1% crystal violet, and viral plaque formation was observed. After gentle rinsing with distilled water and air drying, the number of viral plaques was counted. Figure 2 (A and 2B). The results showed a significant increase in viral plaque formation in the SOD2 knockout group, indicating that gene deletion enhances VCV replication. Western blot analysis further showed that viral protein expression in SOD2 knockout cells was significantly upregulated compared to the control group after 24 hours. These results indicate that SOD2 can effectively inhibit VCV replication. Figure 2 (C and 2D).

[0058] To further validate the antiviral effect of SOD2, A549-SOD2 KO cells and control cells were infected with GFP-expressing VCV at graded infection folds (MOI). Viral spread was controlled by covering the cells with 1% methylcellulose, and the formation of fluorescent plaques was monitored by fluorescence microscopy. SOD2-KO cells exhibited significantly larger viral plaques than the control group. Figure 2 E and 2F) indicate enhanced intercellular propagation following SOD2 depletion. Plaque diameter was quantified using ImageJ (E and 2F). Figure 2 (G and 2H). At low infection folds (MOI=0.1), SOD2-KO cells showed an increase in plaque number and diameter compared to the control group. In contrast, at high MOI (MOI=0.5), plaque diameter in KO cells still increased, but the number of plaques decreased. This suggests that SOD2 knockout accelerates plaque expansion, enhances intercellular viral transmission, and leads to the fusion of adjacent plaques to form larger plaques, resulting in a reduction in plaque number.

[0059] 2.3 SOD2 inhibits acne plaque formation in vivo

[0060] Preliminary in vitro studies have shown that SOD2 can significantly inhibit VCV virus infection. To further investigate whether SOD2 has the same antiviral function in animal models, a classic research animal model—the rabbit—was used in infection experiments. First, a liposome-SOD2 plasmid complex was prepared using transfection reagents, and a control group was set up. This complex was injected intradermally into the hairless skin on the back of the rabbit. 48 h later, VCV (1×10⁻⁶) was inoculated intradermally at the same injection site. 6 PFU), and then the formation and progression of pox spots on the rabbit's back were continuously observed at different infection time points ( Figure 3 A). On day 5 post-infection, control group rabbits showed significant redness and swelling at the injection site, which subsequently developed into ulcerative lesions and gradually scabbed over. Significant scab formation was observed by day 7, after which the scabs gradually subsided, but a raised area remained at the injection site on day 20. In contrast, SOD2-treated rabbits showed only mild redness and swelling on day 5 post-infection, without ulceration. By day 7, no scab-like lesions had formed, and healing had begun. By day 20, the skin damage at the injection site had largely subsided and completely healed. Figure 3 (B) These results indicate that rabbits in the control group developed typical pox lesions and significant skin damage after VCV infection, while rabbits in the SOD2-treated group showed no obvious pox lesions and a significantly reduced degree of skin damage. This phenomenon confirms that SOD2 expression can effectively inhibit the infection process of VCV in vivo.

[0061] In summary, to elucidate the function of SOD2 in antiviral therapy, this invention designed a specific sgRNA targeting exon 2 of the SOD2 gene (NM_000636.4). Using CRISPR-Cas9-mediated gene editing technology combined with a lentiviral delivery system, an SOD2 gene knockout cell line was successfully established. Complete gene deletion was confirmed by DNA sequencing and Western blotting, validating the knockout at both the genomic and protein levels. The SOD2 knockout cell model provides a reliable platform for functional studies. This invention provides a basic framework for exploring the role of SOD2 in antiviral therapy, and the established SOD2 knockout model provides a crucial experimental platform for elucidating SOD2-mediated antiviral defense mechanisms. Furthermore, analyzing the interaction between poxvirus and the host will accelerate the development of targeted antiviral strategies and enhance preparedness against novel threats from poxviruses.

[0062] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An sgRNA, characterized in that, Its sequence is shown in SEQ ID NO.1~SEQ ID NO.

2.

2. An expression carrier, characterized in that, It includes the sgRNA as described in claim 1.

3. A CRISPR / Cas9 system, characterized in that, It includes the sgRNA as described in claim 1 and the Cas9 protein.

4. A method for SOD2 gene knockout, characterized in that, It is used for non-disease treatment purposes, including the step of using the sgRNA as described in claim 1.

5. The SOD2 gene knockout method according to claim 4, characterized in that, It includes the following steps: (1) Primer design: The primer sequences are shown in SEQ ID NO.1~SEQ ID NO.2; (2) Plasmid construction: This includes the step of digesting the vector with the restriction endonuclease BsmBI; (3) Lentiviral packaging and stable strain screening: The plasmid obtained in step (2) was transfected into 293T cells to obtain lentiviral supernatant, which was then used to infect A549 cells. Finally, the cells were screened using culture medium containing puromycin. (4) Cell enzyme digestion and sequencing identification.

6. The SOD2 gene knockout method according to claim 5, characterized in that, The enzyme digestion vector includes LentiCRISPRv2.

7. The application of the sgRNA of claim 1, the expression vector of claim 2, the CRISPR / Cas9 system of claim 3, and / or the SOD2 gene knockout method of any one of claims 4 to 6 in the targeted knockout of the SOD2 gene.

8. An SOD2 knockout cell line, characterized in that, It is prepared by using the sgRNA of claim 1, the expression vector of claim 2, the CRISPR / Cas9 system of claim 3, and / or the SOD2 gene knockout method of any one of claims 4 to 6.

9. The application of the sgRNA of claim 1, the expression vector of claim 2, the CRISPR / Cas9 system of claim 3, the SOD2 gene knockout method of any one of claims 4 to 6, and / or the cell line of claim 7 in SOD2 antiviral research and / or antiviral drug preparation.

10. The application according to claim 9, characterized in that, The virus mentioned includes vaccinia virus.

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