Application of CPA6 gene or its encoded protein in regulating host resistance to bovine enterovirus

Abstract

The present invention discloses the application of the carboxypeptidase 6 (CPA6) gene in regulating host resistance to bovine enterovirus (BEV). Using CRISPR / Cas9 gene editing technology, a bovine kidney cell line with CPA6 gene knockout and a cell model resistant to BEV virus infection were constructed. The results of virological detection showed that the proliferation rate of BEV in gene-knockout cells was significantly lower than that in wild-type cells, while complementing the CPA6 gene caused the antiviral effect of the cells to disappear, indicating that the CPA6 gene plays an important role in regulating host resistance to BEV infection. The present invention identified the function of the CPA6 gene at the cellular level and discovered for the first time that the gene can be used as a potential target for gene-edited animal design in disease-resistant breeding, prevention and treatment of BEV infection, which is of great significance for reducing drug abuse and avoiding epidemics.

Description

Technical Field

[0001] The present invention belongs to the technical field of prevention and treatment of animal infectious diseases, and particularly relates to the application of the CPA6 gene in regulating host resistance to bovine enterovirus (BEV). Background Art

[0002] Bovine enterovirus (BEV), a small RNA virus, is one of the main pathogens causing respiratory and intestinal diseases in cattle. Infection with this virus can cause severe problems in cattle, such as respiratory distress, diarrhea, indigestion, and growth retardation, severely impacting cattle health and productivity. The etiology and ecology of this virus require further investigation, and currently no effective treatment has been established. Therefore, in-depth research on BEV-host interactions and identification of host factors associated with viral infection are crucial for the effective control of BEV infection.

[0003] Currently, much research focuses on epidemiological investigations and the development of diagnostics and vaccines, with relatively little research examining the mechanisms of host-BEV interactions. However, CRISPR / Cas9 high-throughput screening technology has matured and has been used in a variety of mammals, successfully identifying numerous susceptibility genes involved in host-pathogen interactions. Studying the molecular mechanisms of host-pathogen interactions and understanding the specific mechanisms of host-virus interaction can provide target molecules for the development of new vaccines and drugs, as well as for gene editing to create disease-resistant varieties. This provides a theoretical basis for the precise and effective prevention and treatment of BEV infection.

[0004] Carboxypeptidase A6 (CPA6) was discovered in 2002 through bioinformatics research on the human genome. Its primary function is zinc-binding metallocarboxypeptidase activity, which has not been reported in cattle. Studies have shown that missense mutations in the CPA6 gene at Arg36His and Asn271Ser can cause juvenile myoclonic epilepsy; increased methylation of the CPA6 promoter can lead to temporal lobe epilepsy and febrile seizures in patients; CPA6 is significantly upregulated in early-stage oral squamous cell carcinoma and is considered a potential predictive target for active colitis; and CPA6 is involved in the novel glucose-lowering mechanism of metformin. However, the antiviral function of CPA6 has not been reported. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a function of the CPA6 gene, especially its function in regulating host resistance to BEV infection.

[0006] To achieve the above objectives, the present invention utilizes CRISPR / Cas9 gene editing technology to construct a targeting vector for the CPA6 gene in a bovine kidney (Madin-Darby bovine kidney) cell line (MDBK). Through vector transfection and cell screening, a CPA6 knockout cell line and a cell model resistant to BEV infection were established. Virological testing revealed that CPA6 knockout cells exhibited significant resistance to BEV infection. The viral proliferation rate in knockout cells was significantly lower than that in wild-type cells, and viral titers were significantly reduced, indicating that CPA6 knockout effectively inhibited viral infection and proliferation at the cellular level. However, complementation of the CPA6 gene abolished the cellular antiviral effect, suggesting that the CPA6 gene plays an important role in regulating host antiviral capacity. The present invention utilizes CRISPR / Cas9-mediated gene knockout to establish a cell model that effectively resists BEV infection, confirming the role of the CPA6 gene as a target in viral resistance. This strategy can be applied to the preparation of CPA6-modified cells and animals resistant to BEV infection and the development of new drugs.

[0007] A second objective of the present invention is to provide reagents for knocking out or silencing the CPA6 gene for use in the preparation of anti-BEV drugs, animal cells, and species. The reagents include: a Cas9 gene-editing protein or its expression vector, and a sgRNA or its expression vector that directs the Cas9 gene-editing protein to specifically bind to the CPA6 gene. However, the present invention is not limited to reagents developed based on CRISPR / Cas9 technology; other reagents designed for knocking out or silencing the target CPA6 gene using other gene knockout or silencing methods known in the art are also within the scope of the present invention.

[0008] A third objective of the present invention is to provide an anti-BEV drug containing a reagent for knocking out or silencing the CPA6 gene. The reagent includes, but is not limited to, a Cas9 gene-editing protein or its expression vector, and a sgRNA or its expression vector that directs the Cas9 gene-editing protein to specifically bind to the CPA6 gene. The present invention has demonstrated at the cellular level that this drug can effectively knock out or silence the CPA6 gene in host cells, thereby inhibiting viral infection and proliferation. Based on this, it is also possible to use this drug to perform gene editing on other cells or animals, particularly cattle, to achieve the same effect.

[0009] A fourth object of the present invention is to provide a bovine kidney cell line in which the CPA6 gene is knocked out or silenced, which has anti-BEV ability and has application prospects in virus isolation and identification and antiviral host factor research.

[0010] A fifth object of the present invention is to provide a method for preparing a bovine kidney cell line in which the CPA6 gene is knocked out or silenced, the method comprising the following steps:

[0011] (1) Design sgRNA sequences and primers targeting the CPA6 gene and construct sgRNA expression plasmids;

[0012] (2) Lentiviral vector packaging of sgRNA expression plasmid;

[0013] (3) Infect bovine kidney cells containing Cas9 protein with a lentiviral vector containing an sgRNA expression plasmid to obtain a CPA6 knockout polyclonal bovine kidney cell line;

[0014] (4) Select monoclonal cells from the polyclonal bovine kidney cell line.

[0015] The NCBI accession number of the CPA6 gene is GenBank accession no: 100337371, and the encoded protein sequence is shown in SEQ ID NO.1.

[0016] The present invention successfully constructed CPA6 gene knockout bovine kidney cells using the CRISPR / Cas9 gene editing method. The experimental results showed that the cells can resist BEV infection and reduce the viral titer, and the method has high reproducibility.

[0017] The present invention has the following advantages:

[0018] (1) The present invention identified the function of the CPA6 gene at the cellular level through gain-of-function and loss-of-function experiments, respectively. It was found for the first time that this gene can regulate the host's ability to resist BEV infection. It can be used as a potential target for the design of gene-edited animals for the prevention and treatment of cattle anti-BEV infection, which is of great significance for reducing drug abuse and avoiding epidemics.

[0019] (2) The CPA6 gene is expected to be used as a target for drug design to screen candidate compounds for anti-BEV infection. Test compounds are added to the cell model and the expression level of the CPA6 gene in the cells is detected to screen out test compounds that can significantly reduce the expression level of the CPA6 gene.

[0020] (3) The present invention successfully constructed a CPA6 gene knockout MDBK cell line using CRISPR / Cas9 gene editing technology. This method has the advantages of being simple, fast, low-cost, and having high editing efficiency, and can be used to study host factors related to BEV infection.

[0021] For more detailed technical solutions, please refer to the specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1: Schematic diagram of sequencing results of CPA6 knockout MDBK monoclonal cells (CPA6-KO). In the figure: WT is the wild-type MDBK cell sequence, and - represents base deletion.

[0023] Figure 2 : Relative quantitative PCR results of CPA6-knockout MDBK monoclonal cells (CPA6-KO). In the figure: WT is wild-type MDBK, ***p<0.001, mean±SEM (n=3).

[0024] Figure 3 : Results of EdU cell proliferation assay in CPA6-knockout MDBK monoclonal cells (CPA6-KO). In the figure: WT is wild-type MDBK, ns indicates no significant difference, mean ± SEM (n = 3).

[0025] Figure 4 :TCID of CPA6-KO MDBK monoclonal cells against viruses 50 In the figure: WT is wild-type MDBK, **p<0.01, ***p<0.001, mean±SEM (n=3).

[0026] Figure 5 : Absolute quantitative qPCR results of viral resistance of CPA6-knockout MDBK monoclonal cells. In the figure: WT is wild-type MDBK, *p<0.05, ***p<0.001, mean±SEM (n=3).

[0027] Figure 6 : Relative quantitative PCR results of CPA6 gene complemented cells. In the figure: WT is wild-type MDBK, ***p<0.001, mean±SEM (n=3).

[0028] Figure 7 : TCID of BEV amplified by CPA6 gene complemented cells 50 Phenotypic verification: In the figure: WT is wild-type MDBK, ***p<0.001, mean±SEM (n=3). DETAILED DESCRIPTION

[0029] The technical solutions of the present invention are further described in detail below with reference to specific examples. It should be understood that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the claims. Any modifications or equivalent substitutions made by those skilled in the art based on the following examples should be considered within the scope of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally performed according to conventional conditions or reference texts such as the Molecular Cloning Protocol (4th edition), or according to the methods recommended in the manufacturer's manual. Materials whose sources are not specified in the examples, such as MDBK cells, 293T cells, packaging plasmids, and T-vectors, are commonly known in the art and can be constructed independently based on literature reports or obtained commercially. Detailed information on all plasmids used in this experiment can be found at http: / / www.addgene.org / . The restriction enzyme plasmid is pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP (#67991), and the helper plasmids are pMD2.G (#12259) and PSPAX2 (#12260).

[0030] Example 1: Construction of CPA6 gene knockout MDBK monoclonal cell line

[0031] 1. Recovery and Passaging of MDBK Cell Lines Containing Cas9 Protein

[0032] First, remove the MDBK cells (MDBK-Cas9) containing Cas9 protein that were previously stored in this laboratory from liquid nitrogen and quickly place them in a 37°C water bath, shaking continuously during the process to thaw the cells. Place the thawed cells on a sterile operating table and use a pipette to first draw out 5mL of cell culture medium (DMEM containing 5% FBS and 2% double-antibody), then draw out the cell suspension, repeatedly pipette and pour it into a centrifuge tube. Centrifuge at 1000r / min for 5 minutes and discard the supernatant. Take 2mL of cell culture medium and add it to the centrifuge tube. After repeated pipetting, transfer the cells to a six-well plate and culture them in a 37°C, 5% CO2 constant temperature incubator.

[0033] Subculture is performed when the cells are approximately 95% to 100% confluent in a six-well plate. The specific procedure is to remove the six-well plate filled with MDBK-Cas9 cells from the incubator, discard the old culture medium, add 1 mL of PBS to wash, discard the PBS, add 1 mL of trypsin, and incubate in a 37°C, 5% CO2 incubator for 3 minutes. Observe for cell detachment, add an appropriate amount of cell culture medium, and repeatedly pipette and vortex for more than 10 times. Transfer the tube to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant. Add 6 mL of cell culture medium, pipette and mix thoroughly, then transfer the tube to three wells of the six-well plate and return it to the incubator for continued culture.

[0034] 2. Design sgRNA sequence targeting CPA6 gene and construct recombinant plasmid

[0035] 2.1 Design of sgRNA sequence targeting CPA6 gene

[0036] As shown in Table 1, a sgRNA was designed based on the CPA6 gene sequence in NCBI (GenBank accession no: 100337371), and a pair of primers were designed for sgRNA for plasmid construction.

[0037] Table 1 sgRNA sequence and plasmid construction primer sequence information

[0038]

[0039] 2.2 sgRNA plasmid construction

[0040] Annealing: Take 5 μL of each of the forward and reverse primers for the sgRNA and perform the annealing procedure, i.e., denaturation at 95°C for 10 minutes and annealing at 65°C for 60 minutes.

[0041] Ligation of target fragment to T-vector: Then, prepare the reaction system according to Table 2 to carry out the ligation reaction between the target fragment and T-vector (company: Takara; product number: 6013) at a temperature of 25°C for 30 min and 65°C for 10 min.

[0042] Table 2 T4 ligase reaction system

[0043]

[0044] Transformation: Take 50 μL of competent cells melted in an ice bath (Company: Beijing Quanshijin Biotechnology; Product No.: CD201), add the above-mentioned ligation product, gently flick to mix, and place in an ice bath for 30 minutes; heat shock in a 42°C water bath for 45 seconds, and then quickly transfer the tube to ice for 2 minutes. Be careful not to shake the centrifuge tube during this process; add 500 μL of sterile LB medium to each centrifuge tube, mix well, and place in a shaker at 37°C and 200 rpm for 1 hour to allow the bacteria to recover; then centrifuge at 4000 rpm for 5 minutes, discard 350 μL of supernatant, and resuspend the transformed competent cells in the remaining 200 μL, add to the LB agar plate containing ampicillin resistance, spread the cells evenly, and after the liquid is absorbed, invert the plate and culture at 37°C overnight.

[0045] Monoclonal detection: Pick a single clone and add it to LB liquid medium containing ampicillin resistance. Incubate at 37°C, 200 rpm in a shaker for approximately 4-5 hours. Mix thoroughly. Take 200 μL of the culture and send it to Qingke for sequencing.

[0046] Extraction of sgRNA expression plasmid: The sgRNA expression plasmid was extracted from the successfully sequenced monoclonal bacterial solution using a plasmid extraction kit (company: OmegaBio-Tek; product number: D6950-02).

[0047] 3. Obtaining a CPA6-knockout MDBK polyclonal cell line

[0048] 3.1 Lentiviral packaging of sgRNA expression plasmid

[0049] Recovery and culture: Resuscitate 293T cells in a 10 cm cell culture dish and passage them after confluence. Take 293T cells with good growth status of about 3 generations (10 cm cell culture dish, 90%-95% confluence, fresh antibiotic-free DMEM medium containing 10% FBS) for lentiviral packaging.

[0050] Transfection: Before transfection, discard the old culture medium, add DMEM culture medium containing 5% FBS and 1% penicillin-streptomycin double antibody and continue to culture in the incubator (consider washing with PBS if there are many floating cells); for a 10 cm cell culture dish, the total amount of plasmid for lentiviral packaging is 24 μg, of which (PMD2.G: PSPAX2: sgRNA expression plasmid = 1:2:3); add the corresponding volume of plasmid to 500 μL Jetprime Buffer in sequence, gently pipette to mix, add 40 μL JetprimeRegent, gently pipette to mix, let stand at room temperature for 10 minutes, then add to the culture dish, shake gently and return to the incubator; 4-6 hours after virus packaging, change to 10 mL of DMEM culture medium containing 5% FBS and 1% double antibody, return to the incubator and continue culture; 24 hours after the medium change, add 10 mL of DMEM culture medium containing 5% FBS and 1% double antibody, mix well and return to the incubator.

[0051] Ultracentrifugation: After 60-72 hours of virus packaging, observe cell morphology and collect cell supernatant (one 50 mL centrifuge tube for every two 10 cm cell culture dishes). Seal with sealing film and centrifuge at 3000 rpm / min at 4°C for 10 minutes. Take the supernatant and filter it through a 0.45 μm filter into an ultracentrifuge tube. Then, ultracentrifuge at 30,000 rpm at 4°C for 2.5 hours. After that, pour out the supernatant and invert it on absorbent paper to absorb the residual liquid. Add 120 μL of pre-cooled PBS to each tube and resuspend it. After blowing it open and mixing it thoroughly, transfer the same virus concentrate to the same collection tube and disperse it at 4°C overnight. According to the needs of later experiments, aliquot and store it at -80°C.

[0052] 3.2sgRNA lentiviral transfection into MDBK-Cas9 cell line

[0053] MDBK-Cas9 cells were revived in six-well plates and passaged at a 1:3 ratio after confluence. When cells reached approximately 60% confluence, the old culture medium was discarded and 1 mL of cell maintenance medium (DMEM containing 2% FBS and 1% double-antibody) was added to each well. This was followed by 1 μL of polybreme and 40 μL of sgRNA lentivirus, mixed thoroughly, and incubated for 24 hours. The old culture medium was discarded and 2 mL of culture medium containing 5% FBS and 1% double-antibody was added. After another 24 hours of incubation, the cells were replaced with 2 mL of DMEM containing 5% FBS, 1% double-antibody, and 2 μg / mL puromycin for drug selection. After all the negative control drug-screened wells had died, the positive and negative controls were passaged (Note: The positive control wells were passaged at a 1:2 ratio in a six-well plate, and the negative control wells were passaged in two wells, one of which served as the negative control drug selection group). This drug selection process lasted for a total of 7 days, resulting in a CPA6-knockout MDBK polyclonal cell line.

[0054] 4. Selection of CPA6-knockout MDBK monoclonal cells

[0055] CPA6 knockout MDBK polyclonal cells were revived in six-well plates. Once confluent, the cells were counted and 100 cells were plated onto one 96-well plate, with two plates plated for each sgRNA knockout cell line. Cell culture medium was changed every five days (DMEM supplemented with 10% FBS and 1% double-antibody). Cells were observed daily. Cells that had grown to full size in a single well were digested and transferred to a six-well plate. A portion was expanded and frozen, while a portion was used to extract DNA using the TIANamp Genomic DNA Kit.

[0056] The extracted DNA was amplified by PCR. The primer sequences are shown in Table 3. The PCR reaction system was prepared according to Table 4. The program was pre-denaturation at 98°C for 5 minutes, cycle number 1; denaturation at 98°C for 10 seconds, annealing at 57°C for 15 seconds, extension at 72°C for 30 seconds, cycle number 35; and finally extension at 72°C for 5 minutes. The PCR product was sent to Qingke Bio for sequencing and compared with the wild-type MDBK to determine the mutation type. The sequencing results are shown in Figure 4. Figure 1 As shown, the CPA6 knockout MDBK cell line numbered CPA6-KO has an 11-base deletion.

[0057] Table 3 Sequence information of primers for amplifying target fragments

[0058]

[0059] Table 4 PCR amplification reaction system

[0060]

[0061]

[0062] Example 2: Verification of transcriptional levels in CPA6-knockout MDBK monoclonal cells

[0063] RNA was extracted from wild-type MDBK and CPA6 knockout monoclonal cell lines numbered CPA6-KO using the Trizol method, and reverse transcribed into cDNA using a reverse transcription kit (Company: Nanjing Novezan Biotechnology Co., Ltd.; Product No.: R223-01). Relative quantitative qPCR was performed to verify the expression of CPA6 transcription levels. The primer sequences for relative quantitative qPCR are shown in Table 5. The relative quantitative qPCR reaction system was prepared according to Table 6. The program was pre-denaturation at 95°C for 5 minutes, cycle number 1; denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and the instrument default melting curve. The results are shown in Table 5. Figure 2 It was shown that compared with wild-type MDBK cells, the expression level of CPA6 knockout monoclonal cell line numbered CPA6-KO was significantly decreased at the transcriptional level.

[0064] Table 5 Relative quantitative qPCR primer sequence information

[0065]

[0066] Table 6 Relative quantitative qPCR amplification reaction system

[0067]

[0068] Example 3: Verification of EdU cell proliferation levels in CPA6-knockout MDBK monoclonal cells

[0069] To evaluate the cell proliferation level after CPA6 knockout, wild-type MDBK and CPA6-KO cell lines were cultured at 5×10 5 Cells were seeded into 6-well plates. The cells were transferred to DMEM medium supplemented with 10% FBS and 1% double-antibody and incubated in a 37°C and 5% CO2 incubator. After incubation for 24 hours, BeyoClick with Alexa Fluor 555 (Company: Beyotime; Product No.: C0075s) was used to detect the presence of Alexa Fluor 555. TM EdU cell proliferation assay was performed using an EdU cell proliferation kit, and cell nuclei were stained with DAPI (Company: Biyuntian; Product No.: C1005) for 10 minutes at room temperature in the dark. Stained cells were observed using a fluorescence microscope. The ratio of EdU-positive cells was calculated using Image J software. Imaging was performed on three independent wells, and one field of view was randomly captured from each well. The results are shown in Figure 2. Figure 3 The results showed that the proliferation ability of CPA6 gene knockout cells did not change significantly compared with wild-type MDBK cells, and this cell line can be used for subsequent validation experiments.

[0070] Example 4: Using TCID50 Absolute quantitative qPCR detection of the anti-BEV ability of knockout cells

[0071] Wild-type MDBK and knockout cell lines numbered CPA6-KO were revived in T25 cell flasks, and the cells were counted after they were fully grown. 3.5×10 6 The cells were plated in T25 cell flasks and counted after 24 hours of adherence. The cells were then inoculated with BEV at an MOI of 0.1. The culture supernatants and culture supernatant + cell mixtures were collected 12, 24, and 36 hours after inoculation, and the TCID of BEV in the wild-type MDBK and CPA6-KO knockout cell line samples was detected. 50 , and observe their phenotypic differences. Figure 4 It was shown that after infection with BEV, the viral titer of the CPA6 knockout cell line was significantly reduced compared with that of wild-type MDBK.

[0072] Virus RNA extraction kit (Company: Nanjing Novezan Biotechnology Co., Ltd.; Catalog No.: RM402-01) was used to determine the viral TCID 50 Viral RNA was extracted from the virus sample after PCR, and the RNA was reverse transcribed into cDNA using a reverse transcription kit (Company: Nanjing Novezan Biotechnology Co., Ltd.; Product No.: R223-01). The cDNA product was used as a template for absolute quantitative qPCR. The primer sequences for absolute quantitative qPCR are shown in Table 7. The absolute quantitative qPCR reaction system was prepared according to Table 6 above. The amplification program was as follows: pre-denaturation at 95°C for 5 minutes, cycle number 1; denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and the instrument default melting curve. The results are shown in Table 7. Figure 5 Display, same as TCID 50 The assay results were consistent, and the BEV virus copy numbers in the knockout cell lines of CPA6 were significantly reduced compared with those in the wild-type MDBK.

[0073] Table 7 Absolute quantitative qPCR primer sequence information

[0074]

[0075] Example 5: Verification of CPA6 gene complementation

[0076] 1. Construction of CPA6 gene complementation plasmid

[0077] RNA was extracted from wild-type MDBK cells and reverse engineered to obtain cDNA. This cDNA was then used as a template for PCR amplification of the CPA6 target gene using homologous recombination primers configured according to Table 4. A Flag tag was added before the stop codon at the end of the target sequence. The amino acids near the NGG position of the sgRNA were mutated to synonymous amino acids to generate the CPA6 gene complementing sequence. The pLVX-EGFP-IRES-Neo vector was double-digested with EcoRI (New England Biolabs; Catalog No. R0101V) and BamHI (New England Biolabs; Catalog No. R0136V), and the 8269 bp vector band was recovered. The target band and the vector band were ligated using homologous recombination enzyme (Nanjing Novezan Biotechnology Co., Ltd.; Catalog No. C112-01) to construct the pLVX-c CPA6-flag complementing plasmid. The ligation was performed overnight at 4°C, and the cells were identified by picking and sequencing to obtain the pLVX-c CPA6-flag complementing plasmid. Endotoxin-free plasmids were extracted using a plasmid extraction kit (Omega Bio-Tek; Catalog No. D6950-02) and stored at -80°C. The enzyme digestion reaction system for the pLVX-EGFP-IRES-Neo vector is shown in Table 8, and the enzyme digestion and ligation system for the complemented sequence fragment and vector is shown in Table 9.

[0078] Table 8 Enzyme digestion reaction system

[0079]

[0080] Table 9 Replication sequence fragment and vector enzyme digestion ligation system

[0081]

[0082] 2. Obtaining CPA6 complementation cell line

[0083] Refer to the method of step 3.1 of Example 1 to package the lentiviral pLVX-c CPA6-flag complementing plasmid, that is, replace the "sgRNA expression plasmid" in the step with the above-mentioned pLVX-c CPA6-flag complementing plasmid.

[0084] The knockout cell line CPA6-KO was revived in six-well plates and passaged at a 1:3 ratio after confluence. When cells reached approximately 60% confluence, the old culture medium was discarded and 1 mL of cell maintenance medium (DMEM containing 2% FBS and 1% double-antibody) was added to each well. This was followed by 1 μL of polybreme and 40 μL of the CPA6 complementing plasmid lentivirus, mixed thoroughly, and incubated for 24 hours. The old culture medium was discarded and 2 mL of culture medium containing 5% FBS and 1% double-antibody was added. After another 24 hours of incubation, the medium was replaced with 2 mL of DMEM containing 5% FBS, 1% double-antibody, and 400 μg / mL neomycin for drug selection. After all the negative control drug-selected wells died, the positive and negative controls were passaged (Note: The positive control wells were passaged at a 1:2 ratio in six-well plates, and the negative control wells were passaged in two wells, one of which served as the negative control drug selection group). This drug selection process lasted for a total of 7 days, resulting in the CPA6 complemented MDBK cell line. Total RNA of the replenished cells was extracted using Trizol, and the relative quantitative method according to Example 2 was used to verify whether the replenishment experiment was successful. The results are as follows: Figure 6 It showed that the CPA6 gene was successfully complemented.

[0085] 3. Phenotypic Identification of CPA6 Complementary Cell Lines

[0086] Referring to Example 4, wild-type MDBK cells and CPA6 complement cells were infected with BEV virus and TCID 50 To verify whether the phenotype is restored, the results are as follows Figure 7 It was shown that after CPA6 gene complementation, the CPA6 gene transcription level in the complemented cell line was significantly higher than that in the wild-type MDBK, and the complemented cells restored their susceptibility to BEV.

Claims

1. Use of a reagent for knocking out or silencing the CPA6 gene in the preparation of an anti-bovine enterovirus (BEV) drug and animal cells or varieties, the reagent comprising a Cas9 gene-editing protein or its expression vector, and a gRNA or its expression vector that directs the Cas9 gene-editing protein to specifically bind to the CPA6 gene. The amino acid sequence encoded by the CPA6 gene is shown in SEQ ID NO.

1.

2. A BEV-resistant bovine kidney cell line, wherein the bovine kidney cell line has anti-BEV ability due to the knockout or silencing of the CPA6 gene, the lack of expression of the endogenous CPA6 gene or the reduced expression level, and the amino acid sequence encoded by the CPA6 gene is shown in SEQ ID NO.

1.

3. A method for preparing a BEV-resistant bovine kidney cell line, characterized in that The following steps are involved: (1) Design the sgRNA sequence and primers targeting the CPA6 gene and construct the sgRNA expression plasmid; (2) Lentiviral vector packaging of sgRNA expression plasmid; (3) Infect bovine kidney cells containing Cas9 protein with a lentiviral vector containing an sgRNA expression plasmid to obtain a polyclonal bovine kidney cell line in which the CPA6 gene was knocked out; (4) Select monoclonal cells from the polyclonal bovine kidney cell line in which the CPA6 gene was knocked out. The amino acid sequence encoded by the CPA6 gene is shown in SEQ ID NO.

1.

4. The method for preparing the BEV-resistant bovine kidney cell line according to claim 3, wherein: The sgRNA sequence is shown as SEQ ID NO.

2.

5. The method for preparing the BEV-resistant bovine kidney cell line according to claim 4, characterized in that: The primer sequences are shown in SEQ ID NO. 3 and 4.

6. Use of the CPA6 gene in preparing a bovine kidney cell line susceptible to BEV, characterized in that: The CPA6 gene was complemented in bovine kidney cells to obtain a high-expression bovine kidney cell line. The amino acid sequence encoded by the CPA6 gene is shown in SEQ ID NO.1.

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