Application of TRAF3 gene and TRAF3 gene knockout pig kidney cell line in preparation of medicine for preventing and / or treating foot-and-mouth disease

Abstract

The invention belongs to the technical field of cell engineering, and particularly relates to application of a TRAF3 gene and a TRAF3 gene knockout pig kidney cell line in preparation of a medicine for preventing and / or treating foot-and-mouth disease, and the Gene ID of the TRAF3 gene is 100156979. According to the invention, two sgRNAs are designed for a pig TRAF3 gene, the sgRNAs are constructed to a gene editing vector, and PK-15 cells are transfected, so that a TRAF3 gene function-deleted cell line is obtained. Compared with a wild type cell, the knockout cell line can significantly promote the replication of FMDV. It is shown that the TRAF3 gene plays an important regulation role in the host anti-FMDV replication process, and a new drug target is provided for preventing or treating FMDV. The TRAF3 gene knockout cell line disclosed by the invention has great scientific significance on wide application of a gene editing technology, prevention of virus transmission and guidance of a reverse genetic system to optimize vaccine design.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell engineering, and particularly relates to the use of a TRAF3 gene and a TRAF3 gene knockout pig kidney cell line in preparing a drug for preventing and / or treating foot-and-mouth disease. Background Art

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious disease caused by the foot-and-mouth disease virus (FMDV). It commonly affects even-toed ungulates such as pigs, cattle, and sheep. Typical clinical symptoms in affected animals include the formation of blisters, which can even lead to ulcers, on the lips, tongue, mouth, udder, and hooves. Due to its wide host range and severe impact, the World Organization for Animal Health (WOAH) has designated it as a notifiable animal disease. FMDV has seven serotypes: O, A, C, Asia 1 (Asia1), South Africa 1 (SAT1), South Africa 2 (SAT2), and South Africa 3 (SAT3). Each serotype has corresponding subtypes. While there is some cross-protection between subtypes, there is no cross-protection between serotypes, making FMD prevention and control challenging. Epidemiological studies have shown that FMD outbreaks occur frequently in over 100 countries, causing significant economic losses to local livestock production. Therefore, in-depth research on the key host factors that regulate FMDV replication and further exploration of their specific mechanisms of action will not only reveal and clarify the virus replication process in host cells and enrich the complex interaction network map between the virus and the host, but also help to clarify the virus's infection mechanism and provide new targets for the prevention and control of epidemics. Summary of the Invention

[0003] The present invention aims to provide the use of the TRAF3 gene and a TRAF3 knockout porcine kidney cell line in the preparation of a drug for the prevention and / or treatment of foot-and-mouth disease. Compared to wild-type cells, the TRAF3 gene-deficient cell line significantly promotes FMDV replication, demonstrating that the TRAF3 gene plays an important regulatory role in host resistance to FMDV replication and providing a new drug target for the prevention or treatment of FMDV.

[0004] The present invention provides use of the TRAF3 gene in preparing a medicament for preventing and / or treating foot-and-mouth disease. The Gene ID of the TRAF3 gene is 100156979.

[0005] As a preferred solution, the TRAF3 gene is used as a target in the preparation and screening of drugs for preventing and / or treating foot-and-mouth disease.

[0006] The present invention also provides an sgRNA sequence for knocking out the TRAF3 gene, wherein the sgRNA sequence includes an sgRNA sequence for target 1 and an sgRNA sequence for target 2;

[0007] The nucleotide sequence of the sgRNA sequence of target 1 is shown in SEQ ID NO: 1;

[0008] The nucleotide sequence of the sgRNA sequence of target 2 is shown in SEQ ID NO: 2;

[0009] The Gene ID of the TRAF3 gene is 100156979.

[0010] The present invention also provides the use of the sgRNA sequence in constructing a TRAF3 gene knockout cell line.

[0011] The present invention also provides a method for constructing a TRAF3 gene knockout pig kidney cell line, comprising the following steps:

[0012] The sgRNA sequences described above were constructed into the gene editing empty vector to obtain the gene editing vector;

[0013] The gene editing vector is mixed with pig kidney cells and transfected to obtain the TRAF3 gene knockout pig kidney cell line.

[0014] As a preferred embodiment, the gene editing empty vector includes the PX459-puro-MCS vector.

[0015] As a preferred solution, the dosage of the gene editing vector is 1-3 μg / 2×10 5 cells; the density of the pig kidney cells is 2×10 5 Pieces / hole.

[0016] As a preferred solution, the transfection time is 22 to 26 hours.

[0017] The present invention also provides a TRAF3 gene knockout pig kidney cell line obtained by utilizing the construction method.

[0018] The present invention also provides a TRAF3 gene knockout pig kidney cell line obtained by the construction method or an application of the TRAF3 gene knockout pig kidney cell line, comprising at least one of the following:

[0019] Preparation of medicines for preventing and / or treating foot-and-mouth disease;

[0020] Research on the function of TRAF3 gene;

[0021] Study on the molecular mechanism of TRAF3 gene regulating FMDV replication;

[0022] Study on the regulation of TRAF3 gene on the replication of other viruses.

[0023] Beneficial Effects: The present invention provides the use of the TRAF3 gene in the preparation of a drug for the prevention and / or treatment of foot-and-mouth disease. The TRAF3 gene has a Gene ID of 100156979. Two sgRNAs targeting the porcine TRAF3 gene were designed, incorporated into a gene-editing vector, and transfected into PK-15 cells to generate a cell line lacking functional TRAF3. Compared to wild-type cells, this knockout cell line significantly promoted FMDV replication. This indicates that the TRAF3 gene plays an important regulatory role in host resistance to FMDV replication and provides a new drug target for the prevention or treatment of FMDV. The TRAF3 knockout cell line developed in the present invention has significant scientific significance for the widespread application of gene editing technology, preventing viral transmission, and guiding the optimization of vaccine design using reverse genetics systems.

[0024] The present invention carried out FMDV infection experiments in WT and PK-TRAF3-KO cells, and detected the expression of FMDV by Western blotting, RT-qPCR, TCID 50 The effects of endogenous TRAF3 on FMDV replication were comprehensively evaluated using methods such as determination. The results showed that compared with wild-type cells, knockout of the TRAF3 gene in the knockout cell line could significantly promote viral mRNA transcription levels, protein expression levels and viral titers, thereby promoting FMDV replication, indicating that the TRAF3 gene plays an important regulatory role in the host's resistance to FMDV replication. At the same time, a TRAF3 gene knockout cell line can be provided for the treatment of foot-and-mouth disease, which has great scientific significance for the widespread application of gene editing technology, prevention of viral transmission and guidance of reverse genetic system optimization vaccine design. The present invention successfully constructed a pig kidney cell line (PK-15) with a TRAF3 gene knockout using CRISPR / Cas9 gene editing technology, which can lay the foundation for research on TRAF3 gene regulation of FMDV replication, provide ideas for research on TRAF3 gene regulation of other viral replication, and provide new drug targets for the prevention or treatment of FMDV. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0026] Figure 1 This is the gRNA design diagram for the porcine TRAF3 knockout cell line in the examples;

[0027] Figure 2Figure 1 shows the construction and identification of the TRAF3 gene knockout cell line in the examples, where A is a Sanger sequencing analysis of the gene deletion of the TRAF3-KO-1 cell line, B is a Sanger sequencing analysis of the gene deletion of the TRAF3-KO-2 cell line, C is Western blotting to identify the TRAF3 knockout cell line, and 1-8 represent different knockout monoclonal cell lines, respectively;

[0028] Figure 3 Western blotting was used to detect the effect of knockout on FMDV protein levels in the cell line in Experimental Example 1;

[0029] Figure 4 For the relative quantitative detection of FMDV replication in WT and TRAF3-KO cells in Experimental Example 2, P < 0.01 is indicated by **;

[0030] Figure 5 is the TCID in Experiment 3 50 Real-time detection of FMDV viral titers in TRAF3-KO and WT cells, P < 0.01 is indicated by **;

[0031] Note: Attached Figures 2 to 5 PK-TRAF3-KO cells refer to TRAF3-KO cells, and also refer to the TRAF3 gene knockout pig kidney cell line. DETAILED DESCRIPTION

[0032] The present invention provides the use of the TRAF3 gene in preparing a drug for preventing and / or treating foot-and-mouth disease. The Gene ID of the TRAF3 gene is 100156979. As a specific embodiment, the use includes using the TRAF3 gene as a target in the preparation and screening of drugs for preventing and / or treating foot-and-mouth disease.

[0033] The present invention also provides an sgRNA sequence for knocking out the TRAF3 gene, wherein the sgRNA sequence includes an sgRNA sequence for target 1 and an sgRNA sequence for target 2;

[0034] The nucleotide sequence of the sgRNA sequence of target 1 is shown in SEQ ID NO: 1: 5'-GGTCAAAAGGTGGACGCGGC-3';

[0035] The nucleotide sequence of the sgRNA sequence of target 2 is shown in SEQ ID NO: 2: 5'-CTACAAGGAACGGTTCGTGA-3';

[0036] The Gene ID of the TRAF3 gene is 100156979.

[0037] The present invention also provides the use of the sgRNA sequence in constructing a TRAF3 gene knockout cell line.

[0038] The present invention also provides a method for constructing a TRAF3 gene knockout pig kidney cell line, comprising the following steps: constructing the above-mentioned sgRNA sequences into a gene editing empty vector to obtain a gene editing vector; mixing the gene editing vector with pig kidney cells and transfecting them to obtain the TRAF3 gene knockout pig kidney cell line. As a specific embodiment, the gene editing empty vector includes a PX459-puro-MCS vector. The dosage of the gene editing vector of the present invention is 1 to 3 μg / 2×10 5 As a specific embodiment, the amount of the gene editing vector can be 1 μg / 2×10 5 cells, 2 μg / 2×10 5 cells and 3 μg / 2×10 5 cells and values ​​between any two values; the density of the pig kidney cells is 2×10 5 The transfection time of the present invention is 22 to 26 hours. As a specific embodiment, the transfection time can be 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, or any value between two values.

[0039] In an embodiment of the present invention, CRISPR / Cas9 gene editing technology was used to successfully construct a pig kidney cell line (PK-15) with TRAF3 gene knockout. The present invention successfully constructed a recombinant plasmid PX459-TRAF3-sgRNA. After transfecting PK-15 cells, puromycin pressure screening was used to eliminate a large number of PX459-TRAF3-sgRNA negative cells. Monoclonal cell lines were obtained by limiting dilution subcloning, thereby improving the knockout efficiency of the target gene in the candidate cell line. Through DNA sequencing and protein level identification, a TRAF3 gene knockout cell line was successfully obtained, laying a certain foundation for in-depth research on the viral infection mechanism of the TRAF3 gene knockout cell line. At the same time, the efficient editing of the target gene was achieved by CRISPR / Cas9 gene editing technology, which can be further promoted and extended to TRAF3 gene knockout in other animal cells, constructing a gene knockout cell line with enhanced TRAF3 antigen expression, and promoting the development of gene function research, disease model construction and precision medicine.

[0040] The present invention also provides a TRAF3 gene knockout pig kidney cell line obtained by utilizing the construction method.

[0041] The present invention also provides a TRAF3 gene knockout pig kidney cell line obtained by the construction method or an application of the TRAF3 gene knockout pig kidney cell line, comprising at least one of the following:

[0042] Preparation of medicines for preventing and / or treating foot-and-mouth disease;

[0043] Research on the function of TRAF3 gene;

[0044] Study on the molecular mechanism of TRAF3 gene regulating FMDV replication;

[0045] Study on the regulation of TRAF3 gene on the replication of other viruses.

[0046] The present invention shows that the FMDV infection test was carried out in WT and PK-TRAF3-KO cells, and the expression of FMDV was detected by Western blotting, RT-qPCR, TCID 50 The effects of the endogenous TRAF3 gene on FMDV replication were comprehensively evaluated using methods such as assays. The results demonstrated that, compared with wild-type cells, knockout of the TRAF3 gene in the knockout cell lines significantly promoted viral mRNA transcription, protein expression, and viral titer, thereby promoting FMDV replication. This suggests that the TRAF3 gene plays an important regulatory role in the host's resistance to FMDV replication. Furthermore, this invention can provide a TRAF3 gene knockout cell line for the treatment of foot-and-mouth disease, which has significant scientific significance for the widespread application of gene editing technology, the prevention of viral transmission, and the guidance of reverse genetics systems for optimizing vaccine design.

[0047] To further illustrate the present invention, the use of the TRAF3 gene and the TRAF3 gene knockout pig kidney cell line provided by the present invention in the preparation of drugs for preventing and / or treating foot-and-mouth disease is described in detail below in conjunction with the examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0048] Unless otherwise specified, the present invention has no special requirements for the raw materials, and commercially available products known to those skilled in the art can be used.

[0049] Vectors, cells and viruses of the present invention: PK-15 cells, BHK-21 cells, FMDV (O / BY / CHA / 2010), PX459-puro-MCS vector (Ran Qiandong, Yang Fan, Zhang Wei, et al. Establishment and application of CRISPR / Cas9-mediated KLHL34 gene knockout cell line [J]. Chinese Journal of Veterinary Science, 2023, 53(7): 851-857. DOI: 10.16656 / j.issn.1673-4696.2023.0129.).

[0050] Reagents and antibodies of the present invention: DMEM cell culture medium and 0.25% EDTA trypsin were purchased from Gibco. Escherichia coli DH5α competent cells and T4 DNA ligase were purchased from Takara Biotechnology (Dalian) Co., Ltd. Fetal bovine serum (FBS) was purchased from Biological Industries (BI). Protein pre-stained markers, Trizol reagent, etc. were purchased from Invitrogen. Western blotting antibody diluent was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. 10×LATaq enzyme was purchased from Kangwei Century Biotechnology Co., Ltd. Prime ScriptTM RT Reagent Kit with gDNAEraser reverse transcription kit and RT-qPCR kit TB GreenTM Premix ExTaqTM II were purchased from Nanjing Novozymes Biotechnology Co., Ltd. Gel recovery kit and plasmid extraction kit were purchased from OMEGA. Jet PRIME Transfection Reagent was purchased from Polyplus Transfection. Protein detection colorimetric kit (PierceTM ECL Western Blotting Substrate) was purchased from ThermoFisher Scientific. Restriction endonuclease Bbs I was purchased from New England Biolabs. Rabbit anti-TRAF3 polyclonal antibody and mouse anti-β-actin antibody were purchased from Proteintech. Cell micro-DNA extraction kit was purchased from Omega. Rabbit anti-FMDV polyclonal antibody (Ran Qiandong, Yang Fan, Zhang Wei, et al. Establishment and application of CRISPR / Cas9-mediated KLHL34 gene knockout cell line [J]. Chinese Journal of Veterinary Science, 2023, 53(7): 851-857. DOI: 10.16656 / j.issn.1673-4696.2023.0129.).

[0051] Statistical analysis of the present invention: GraphPad Prism software was used for statistical analysis and plotting. P < 0.05 is indicated by *, P < 0.01 is indicated by **, P < 0.001 is indicated by ***, and P < 0.0001 is indicated by ****.

[0052] In the present invention, PK-TRAF3-KO cells refer to TRAF3-KO cells, and also refer to TRAF3 gene knockout pig kidney cell lines.

[0053] Example Construction and identification of TRAF3 gene knockout pig kidney cell line

[0054] 1. Design and synthesis of sgRNA primers

[0055] According to the NCBI database query TRAF3 gene sequence (Gene ID: 100156979), and CRISPR / Cas9 design principles, and based on the recommendations of Zhang Feng Laboratory website (http: / / crispr.mit.edu / ), two sgRNA sequences were designed at positions 358 and 463 of the exon segment of the TRAF3 gene sequence ( Figure 1 ). At position 358 of the exon segment, the nucleotide sequence of the sgRNA sequence for target 1 is shown in SEQ ID NO: 1: 5'-GGTCAAAAGGTGGACGCGGC-3'; at position 463 of the exon segment, the nucleotide sequence of the sgRNA sequence for target 2 is shown in SEQ ID NO: 2: 5'-CTACAAGGAACGGTTCGTGA-3'.

[0056] 2. Construction of the recombinant plasmid PX459-TRAF3-sgRNA

[0057] Dilute the upstream and downstream primers of the sgRNA sequence to 10 μmol / μL. Prepare a 50 μL reaction mixture: 22.5 μL of upstream primer F (10 μmol / μL), 22.5 μL of downstream primer R (10 μmol / μL), and 5 μL of 10× PCR buffer. Gently mix. Anneal at 99°C for 5 min. After annealing, let stand at room temperature to form double-stranded DNA. Digest the PX459-puro-MCS vector with Bbs I endonuclease. After electrophoresis on a 1% agarose gel, recover the digested fragment. Ligate the linearized digestion product with the sgRNA sequence using T4 ligase. Transform the ligation product into DH5α competent cells, plate it onto a LB solid culture dish containing Amp, and incubate it inverted at 37°C for 14 h. Select single colonies of uniform size for expansion, and then extract the plasmid and send it to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The positive plasmids were named PX459-TRAF3-sgRNA-1 and PX459-TRAF3-sgRNA-2.

[0058] 3. Cell Transfection and Screening

[0059] PK-15 cells were placed in a 37°C, 5% CO2 incubator to recover and passage. After passage three times, the cell status was observed. When the cell shape was stable and the growth was good, 2×10 cells were added to each well. 5Cells were seeded into 6-well plates at a density of 100 μg and cultured in an incubator to a cell density of approximately 70%. They were then transfected with 2 μg of each of the two (PX459-TRAF3-sgRNA-1 and PX459-TRAF3-sgRNA-2) using jet-PRIME transfection reagent. 24 hours after transfection, cells were digested and seeded into 6-well plates at a 1:3 passage ratio. 4, 5, and 6 μL of puromycin (3 μg / mL) were added, respectively. The cells were then cultured in an incubator for 3 days to screen for sgRNA-positive cells. Fresh DMEM medium was replaced in the 6-well plates, and positive cells were allowed to recover for 1 day. Subcloning was then performed using the limiting dilution method and seeded into 96-well plates with 0.1 mL of DMEM per well, yielding 1 cell per well. The cells were cultured in an incubator for another 7 days. Select wells with monoclonal cells in good condition, digest and transfer them to 48-well plates, add 0.3 mL of DMEM per well, and continue to culture the cells in an incubator for 5 days. After the cells are confluent, transfer them to 12-well plates and 6-well plates for expansion culture.

[0060] 4. DNA Sequencing and Western Blotting

[0061] Wild-type cells (WT) and monoclonal cells to be identified were taken, and DNA was extracted separately according to the instructions of the cell micro-DNA extraction kit. After amplifying the fragment containing the sgRNA targeting site using TRAF3 gene identification primers [F: 5'-TGAGTGGAGTG GAGACTGC-3' (SEQ ID NO: 3); R: 5'-ACTCTGCGATGTCCTATCT-3' (SEQ ID NO: 4)], the plasmid size was identified using 1% agarose gel, the amplified product was recovered from the gel, and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results of wild-type cells and monoclonal cells to be identified were aligned, and cells with base deletion and cells without base deletion were selected and named PK-TRAF3-KO and PK-TRAF3-WT, respectively. The results are shown in Figure 2. Figure 2 As shown in Figures A and B, PK-TRAF3-KO-1 has a base insertion in the sgRN A-1 exon region of the TRAF3 gene, and PK-TRAF3-KO-2 has a base deletion in the sgRN A-2 exon region of the TRAF3 gene. Western blotting experiments were performed on wild-type cells (WT) and monoclonal cells to be identified. It was found that no endogenous TRAF3 protein was detected in multiple monoclonal cell lines ( Figure 2 The above results indicate that the PK-15 cell line with TRAF3 gene knockout (i.e., TRAF3 gene knockout pig kidney cell line) was successfully constructed.

[0062] Experimental Example 1 Effect of TRAF3 gene knockout on foot-and-mouth disease virus protein expression

[0063] To investigate the effect of knockout of the TRAF3 gene on FMDV protein levels, PK-TRAF3-KO and PK-TRAF3-WT cells were seeded into 35 mm culture dishes. After reaching over 90% cell mass, they were infected with FMDV (MOI = 1) and incubated at 37°C. DMEM was removed at various time points (0, 4, 8, and 12 hours). Loading buffer was added and mixed thoroughly. The cells were incubated in a metal bath at 100°C for 10 minutes and allowed to cool to room temperature. Protein samples were subjected to SDS-PAGE electrophoresis and transferred to the membrane at 100V in an ice-water bath for 2 hours. Blocking was performed with 5% skim milk powder for 1 hour at room temperature. The membranes were incubated with the corresponding primary antibody overnight at 4°C. The membranes were washed three times with TBST and incubated with the corresponding secondary antibody for 1 hour at room temperature. Western blotting analysis was performed.

[0064] TRAF3 gene knockout PK-15 cell line (PK-TRAF3-KO) and wild-type PK-15 cells (WT) were simultaneously infected with FMDV and samples were collected at the same time point. Western blotting was used to detect the protein expression of TRAF3 and FMDV. The results showed that knockout of the TRAF3 gene promoted the expression of FMDV protein level ( Figure 3 ).

[0065] Experimental Example 2 Effect of TRAF3 gene knockout on FMDV mRNA levels

[0066] PK-TRAF3-WT and PK-TRAF3-KO cells were seeded into 35 mm culture dishes. After reaching over 90% cell mass, they were infected with FMDV (MOI = 1) and cultured at 37°C. At different time points (8 and 12 hours), the DMEM was discarded and total RNA was extracted using the Trizol lysis method. After concentration determination, a 20 μL reverse transcription reaction system was prepared: 12 μL of ddH2O, 4 μL of 5× HiScript II qRT SuperMix II, and 4 μL of RNA solution diluted in ddH2O. cDNA was amplified. The reverse transcription reaction protocol was: 50°C for 15 minutes, 85°C for 5 seconds. A 10 μL quantitative reaction system was prepared: 3.6 μL ddH2O, 5 μL TB Green PremixEx Taq (Tli RNaseH Plus), 0.2 μL each of the upstream and downstream primers for FMDV relative quantitative analysis [(FMDV-F: 5'-CACTGGTGACAGGCTAAGG-3' (SEQ ID NO: 5), FMDV-R: 5'-CCC TTCTCAGATTCCGAGT-3' (SEQ ID NO: 6)], and 1 μL cDNA. The fluorescence quantitative PCR reaction program was: 95°C for 3 min, 95°C for 10 s, and 60°C for 34 s, for a total of 40 cycles; GAPDH was used as an internal reference gene to perform relative quantification of FMDV mRNA levels. All experiments were repeated 3 times, using 2 -△△CT Methods Analytical data.

[0067] TRAF3 gene knockout PK-15 cell line (PK-TRAF3-KO) and wild-type PK-15 cells (WT) were infected with FMDV, and samples were collected at the same time point. RNA was extracted and reverse transcription was performed, and the relative expression level of FMDV mRNA was detected by RT-qPCR. Figure 4 As shown in Table 1 , the relative expression level of mRNA in PK-TRAF3-KO cells increased significantly after infection with FMDV, indicating that knockout of the TRAF3 gene significantly promoted the FMDV mRNA level.

[0068] Table 1 Relative quantitative detection of FMDV replication in WT and TRAF3-KO cells

[0069] time PK-WT PK-TRAF3-KO 8h 5000 / 5100 8400 / 8500 12h 12000 / 11000 20900 / 21000

[0070] Experimental Example 3 Effect of TRAF3 gene knockout on viral titer

[0071] PK-TRAF3-WT and PK-TRAF3-KO cells were inoculated into 35 mm cell culture dishes, and infected with FMDV (MOI = 1) after the cells grew to more than 90%. They were then placed in a 37°C incubator and cultured. Virus fluid was collected at different time points (24 h and 36 h), frozen and thawed three times, and centrifuged at 12000 rpm at 4°C for 5 min. The supernatant was collected to detect the virus titer. The samples to be tested were incubated for 10 min in serum-free and antibiotic-free DMEM medium. -2 -10 -8 The virus solution was serially diluted at each dilution and inoculated into a confluent monolayer of BHK-21 cells in a 96-well plate. Eight wells were inoculated at each dilution, with 100 μL per well. The cells were cultured in an incubator for 3 days, during which the cytopathic effect (CPE) was observed and recorded. The TCID was calculated according to the Reed-Muench method. 50 .

[0072] TRAF3 gene knockout PK-15 cell line (PK-TRAF3-KO) and wild-type PK-15 cells (WT) were infected with FMDV, and the virus supernatant was collected at the same time point, frozen and thawed three times, and then centrifuged to detect TCID 50 The results are as follows Figure 5 As shown in Table 2, the viral titer of PK-TRAF3-KO cells increased significantly after infection with FMDV, indicating that the functional loss of the TRAF3 gene significantly promoted the FMDV viral titer.

[0073] Table 2 TCID 50 Real-time detection of FMDV viral titers in TRAF3-KO and WT cells

[0074] time PK-WT PK-TRAF3-KO 24h 3.2 / 3 4.6 / 4.8 36h 4.5 / 4.6 6.6 / 6.5

[0075] Thus, the present invention designed two sgRNAs targeting the porcine TRAF3 gene, constructed the sgRNAs into a gene-editing vector, and transfected PK-15 cells to generate a cell line lacking functional TRAF3. Compared to wild-type cells, this knockout cell line significantly promoted FMDV replication. This suggests that the TRAF3 gene plays an important regulatory role in the host's resistance to FMDV replication and provides a new drug target for the prevention or treatment of FMDV. Furthermore, the present invention provides a TRAF3 knockout cell line, which has significant scientific significance for the widespread application of gene editing technology, preventing viral transmission, and guiding the optimization of reverse genetics systems for vaccine design.

[0076] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of the TRAF3 gene in the preparation of a drug for preventing and / or treating foot-and-mouth disease, characterized in that: The Gene ID of the TRAF3 gene is 100156979.

2. The use according to claim 1, characterized in that The invention includes the preparation and screening of drugs for preventing and / or treating foot-and-mouth disease using the TRAF3 gene as a target.

3. The sgRNA sequence for knocking out the TRAF3 gene is characterized by: The sgRNA sequence includes the sgRNA sequence of target 1 and the sgRNA sequence of target 2; The nucleotide sequence of the sgRNA sequence of target 1 is shown in SEQ ID NO: 1; The nucleotide sequence of the sgRNA sequence of target 2 is shown in SEQ ID NO: 2; The Gene ID of the TRAF3 gene is 100156979.

4. Use of the sgRNA sequence according to claim 3 in constructing a TRAF3 gene knockout cell line.

5. A method for constructing a TRAF3 gene knockout pig kidney cell line, characterized in that: The following steps are involved: constructing the sgRNA sequences described in claim 3 into gene editing empty vectors to obtain gene editing vectors; The gene editing vector is mixed with pig kidney cells and transfected to obtain the TRAF3 gene knockout pig kidney cell line.

6. The construction method according to claim 5, characterized in that: The gene editing empty vector includes the PX459-puro-MCS vector.

7. The construction method according to claim 5, characterized in that: The dosage of the gene editing vector is 1-3 μg / 2×10 5 cells; the density of the pig kidney cells is 2×10 5 Pieces / hole.

8. The construction method according to claim 5, characterized in that: The transfection time is 22 to 26 hours.

9. A TRAF3 gene knockout pig kidney cell line obtained by the construction method according to any one of claims 5 to 8.

10. Use of the TRAF3 gene knockout pig kidney cell line obtained by the construction method according to any one of claims 5 to 8 or the TRAF3 gene knockout pig kidney cell line according to claim 9, characterized in that: Include at least one of the following: Preparation of medicines for preventing and / or treating foot-and-mouth disease; Research on the function of TRAF3 gene; Study on the molecular mechanism of TRAF3 gene regulating FMDV replication; Study on the regulation of TRAF3 gene on the replication of other viruses.

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