Application of SELENON gene or encoded protein thereof in regulation and control of host anti-BPIV-3

Abstract

The invention discloses an application of an SELENON gene in regulating and controlling the resistance of a host to a bovine parainfluenza virus type 3 (BPIV (bovine parainfluenza virus)-3), and particularly discloses an application of the SELENON gene to the regulation and control of the host to the BPIV-3. A CRISPR / Cas9 gene editing technology is utilized to construct an SELENON gene knockout MDBK cell line and an anti-BPIV-3 infection cell model, and it is found that gene knockout cells show remarkable resistance to BPIV-3 infection, the proliferation speed of BPIV-3 on the gene knockout cells is remarkably lower than that of wild cells, and the antiviral effect of the cells disappears if the SELENON gene is replenished. The results show that the SELENON gene plays an important role in regulating and controlling the host virus resistance. The antiviral regulation function of the SELENON gene is identified from the cellular level, it is found for the first time that the gene can serve as a potential target for gene editing cells and animal design to be applied to disease-resistant breeding and research and development of bovine anti-BPIV-3 infection prevention and treatment drugs, and great significance is achieved for reducing drug abuse and avoiding epidemic situations.

Description

Technical Field

[0001] The invention belongs to the technical field of animal infectious disease prevention and treatment, and relates to the application of SELENON gene in regulating host resistance to bovine parainfluenza virus type 3 (BPIV-3). Background Art

[0002] Bovine parainfluenza virus type 3 (BPIV-3) is an important pathogen that can cause respiratory diseases in cattle. It belongs to the Paramyxoviridae family, Respirovirus genus. It is an enveloped, non-segmented, single-stranded negative-sense RNA virus with a total length of about 15,000 bp. Bovine parainfluenza caused by BPIV-3 is a contact, febrile, acute respiratory infectious disease in cattle, with fever, cough, general weakness, and depression as the main symptoms. It is also often manifested as dyspnea, abortion or reproductive disorders in cows. Severe cases can lead to death. The pathogen is often mixed with bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), Pasteurella, Mannheimia, mycoplasma and other pathogens, namely Bovine respiratory disease complex (BRDC), commonly known as "transport fever". The main symptoms of sick cattle are high fever, purulent conjunctivitis, tearing, runny nose, frequent coughing, difficulty breathing, and foamy saliva. my country currently lacks effective drugs and vaccines to deal with BPIV-3, and it is particularly important to develop vaccines and drugs that can more effectively prevent and treat the disease.

[0003] Most previous studies used RNA interference (RNAi) library methods for genetic screening, but this method has disadvantages such as low inhibition efficiency and high off-target rate. As the third-generation gene editing technology, CRISPR / Cas9 technology has the advantages of being economical, simple to operate, accurate and efficient. It has been successfully applied in whole genome screening of many mammals such as pigs, humans, mice, and African green monkeys.

[0004] Based on this, the present invention uses CRISPR / Cas9 technology to establish a bovine whole genome cell mutant library, and uses it to screen bovine parainfluenza virus type 3 susceptibility genes, providing technical support for the further development of vaccines and drugs that can more effectively resist BPIV-3. Through whole genome screening of cattle, the present invention found that the selenoprotein N (SELENON) gene or its encoded protein plays an important role in regulating host resistance to BPIV-3 infection. Summary of the invention

[0005] A first object of the present invention is to screen for host factors involved in BPIV-3 replication.

[0006] To achieve the above objectives, the applicant used CRISPR / Cas9 technology to construct a whole genome mutant library of bovine kidney cells (Madin-Darby bovine kidney, MDBK), infected the mutant library with BPIV-3, and then performed high-throughput sequencing to find enriched genes and verify them. The specific implementation plan is as follows:

[0007] First, the monoclonal cell line of MDBK cells containing Cas9 protein selected by the Ruminant Pathogens Branch of the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, was revived and cultured, and the sgRNA plasmid library constructed in the laboratory was packaged into lentivirus. The lentivirus packaged with the sgRNA plasmid library was used to infect MDBK-Cas9 cells. After puromycin screening, the cell mutant library was subjected to high-throughput sequencing, and it was found that the gene coverage reached 99.62% and the sgRNA coverage reached 92.12%, which met the screening requirements. Then, the cell mutant library was infected with BPIV-3 and four rounds of screening were performed. The surviving cells after infection were collected and high-throughput sequencing was performed to screen out host factors that can affect BPIV-3 replication. Finally, single gene deletion cell lines were constructed for these candidate genes, infected with BPIV-3, and the BPIV-3 genome copy number was absolutely quantified. It was found that the deletion of these candidate genes significantly inhibited the replication of BPIV-3, and the antiviral effect of the cells disappeared after the gene was complemented.

[0008] The above results show that the use of cell whole genome mutant library can reveal a group of unknown host key factors involved in BPIV-3 replication, thereby providing new targets for the development of vaccines and drugs against BPIV-3 infection. Therefore, this cell mutant library has extremely high application value. In addition, this cell whole genome mutant library also has the following advantages:

[0009] 1) The designed sgRNA library contains 23,265 protein-coding genes, almost all the annotated protein-coding genes in the bovine genome, with a wide coverage. In addition, four sgRNAs were designed for each gene, targeting the upstream position of the gene open reading frame to ensure the knockout efficiency of the gene. The maximum allowed mismatch base number for each sgRNA is 5bp, reducing the risk of off-target.

[0010] 2) The cell mutant library can be applied to a variety of viruses. Any virus that can replicate and proliferate in MDBK cells and induce cell death can be screened with it. The sgRNA plasmid library can be passaged multiple times, and the constructed cell mutant library can also be passaged in a limited manner, which can ensure that the sgRNA content and coverage will not have a large deviation.

[0011] The second object of the present invention is to provide the SELENON gene screened by the above method in regulating host immunity. Function of BPIV-3.

[0012] To achieve the above purpose, the present invention uses CRISPR / Cas9 gene editing technology to construct a targeting vector targeting the SELENON gene of MDBK cells, and establishes a cell line with SELENON gene knockout and a cell model against BPIV-3 virus infection through vector transfection and cell screening. Virological detection results show that SELENON gene knockout cells show significant resistance to BPIV-3 infection, and the proliferation rate of the virus on gene knockout cells is significantly lower than that of wild-type cells, indicating that at the cellular level, SELENON gene knockout effectively inhibits viral infection and proliferation, while complementing the SELENON gene causes the antiviral effect of the cell to disappear, indicating that the SELENON gene plays an important role in participating in BPIV-3 replication and regulating the host's antiviral ability. The present invention uses CRISPR / Cas9-mediated gene knockout method to establish a cell model that effectively resists BPIV-3 infection, confirming the role of SELENON gene as a target in antiviral infection. This strategy can be applied to the preparation of SELENON gene-modified anti-BPIV-3 infected cells and animals and the development of new drugs.

[0013] The third object of the present invention is to provide a reagent for knocking out or silencing the SELENON gene in the preparation of an antibody. BPIV-3 pharmaceuticals and uses in animal cells and species.

[0014] To achieve the above purpose, the present invention uses CRISPR / Cas9 gene editing technology to knock out the SELENON gene of MDBK cells, and it is found that the gene knockout cells show significant resistance to BPIV-3 infection, and the virus titer is significantly reduced. Therefore, the CRISPR / Cas9 reagent for knocking out the SELENON gene can be used to prepare anti-BPIV-3 drugs and prepare animal cells and varieties that are resistant to BPIV-3 infection. Further, the CRISPR / Cas9 reagent includes: Cas9 gene editing protein or its expression vector, and sgRNA or its expression vector that guides the Cas9 gene editing protein to specifically bind to the SELENON gene. However, the present invention is not limited to reagents developed based on CRISPR / Cas9 technology, and other reagents for knocking out and silencing the target SELENON gene designed using other gene knockout or silencing methods known in the art are also within the scope of protection of the present invention.

[0015] The fourth object of the present invention is to provide a drug against BPIV-3.

[0016] The drug contains a reagent for knocking out or silencing the SELENON gene. Furthermore, the reagent includes: a Cas9 gene editing protein or its expression vector, and a gRNA or its expression vector that guides the Cas9 gene editing protein to specifically bind to the SELENON gene, and is not limited thereto. The present invention has verified at the cellular level that the drug can effectively knock out or silence the SELENON gene of the host cell, thereby inhibiting the infection and proliferation of the virus. On this basis, it is also possible to use the drug to perform gene editing on other cells or animals, especially cattle, and achieve the same function.

[0017] The fifth object of the present invention is to provide a bovine kidney cell line in which the SELENON gene is knocked out or silenced. The bovine kidney cells have the ability to resist BPIV-3 and have application prospects in virus isolation and identification and antiviral host factor research.

[0018] The sixth object of the present invention is to provide a method for preparing a bovine kidney cell line in which the SELENON gene is knocked out or silenced. Preparation method. The method comprises the following steps:

[0019] (1) Design sgRNA sequences and primers targeting the SELENON gene and construct sgRNA expression plasmids;

[0020] (2) Lentiviral vector packaging of sgRNA expression plasmid;

[0021] (3) using a lentiviral vector containing an sgRNA expression plasmid to infect bovine kidney cells containing the Cas9 protein, and obtaining a polyclonal bovine kidney cell line in which the SELENON gene was knocked out;

[0022] (4) Selecting monoclonal cells from a polyclonal bovine kidney cell line.

[0023] The NCBI accession number of the SELENON gene is GenBank accession no: 616379, and the encoded protein sequence is shown in SEQ ID NO.1.

[0024] The present invention successfully constructed SELENON gene knockout bovine kidney cells using the CRISPR / Cas9 gene editing method. The experimental results showed that the cells can resist BPIV-3 infection, inhibit the titer and expression level of the virus, and the method has high reproducibility.

[0025] The present invention has the following advantages:

[0026] 1) The present invention identified the function of the SELENON gene at the cellular level through gain-of-function and loss-of-function experiments, and discovered for the first time that the gene can regulate the host's ability to resist BPIV-3 infection. It can be used as a potential target for the design of gene-edited animals for the prevention and treatment of cattle anti-BPIV-3 infection, which is of great significance for reducing drug abuse and avoiding epidemics.

[0027] 2) The SELENON 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 SELENON gene in the cells is detected to screen out test compounds that can significantly reduce the expression level of the SELENON gene.

[0028] 3) The present invention successfully constructed a SELENON 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 BPIV-3 infection.

[0029] For more detailed technical solutions, please refer to the specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 :Statistical analysis of sequencing of the MDBK cell mutant library. A. MAGeCK analysis of sequencing results of the cell mutant library; B. The number of sgRNAs for each gene in the cell mutant library.

[0031] Figure 2 :Enriched gene analysis of the third and fourth rounds of screening results. A. KEGG and GO analysis of the top 50 genes in the third and fourth rounds of screening; B. KEGG and GO analysis of the top 100 genes in the third and fourth rounds of screening. BPIV-3_3rd: The third round of screening results of the cell mutant library using BPIV-3; BPIV-3_4th: The fourth round of screening results of the cell mutant library using BPIV-3.

[0032] Figure 3 : Genotypes of the SELENON gene single deletion clone cell line. WT: wild-type MDBK; “-” represents the deleted base.

[0033] Figure 4 : Results of relative quantitative PCR of genes after SELENON gene knockout. ****p<0.0001, mean±SEM (n=3).

[0034] Figure 5 : Verification results of EdU cell proliferation level in SELENON gene knockout cells. WT: wild-type MDBK, ns: no significant difference, mean±SEM (n=3).

[0035] Figure 6 :TCID of SELENON gene-deficient cells against viruses 50 Validation. WT: wild type MDBK, ns: no significant difference, **p<0.01, mean±SEM (n=3).

[0036] Figure 7 : Absolute quantitative qPCR validation of SELENON gene-deficient cells' resistance to viruses. WT: wild-type MDBK, ***p<0.001, mean±SEM (n=3).

[0037] Figure 8 : Results of relative quantitative PCR validation of SELENON gene complementation. WT: wild-type MDBK, ****p<0.0001, mean±SEM (n=3).

[0038] Fig. 9 :TCID of BPIV-3 amplified by SELENON gene complemented cells 50 Phenotypic verification. WT: wild type MDBK, ns: no significant difference, **p<0.01, ***p<0.001, mean±SEM (n=3). DETAILED DESCRIPTION

[0039] The technical scheme of the present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. Various modifications or equivalent substitutions made by those skilled in the art on the basis of the following examples should also be deemed to fall within the scope of protection of the present invention. The experimental methods for which specific conditions are not indicated in the following examples are usually implemented according to conventional conditions or reference books such as "Molecular Cloning Experiment Guide" (4th edition), or implemented according to the methods recommended in the operating manual provided by the manufacturer. Materials such as MDBK cells, 293T cells, packaging plasmids, T vectors, etc., whose sources are not indicated in the examples, are all commonly used materials well known in the art and can be constructed by themselves according to literature reports or obtained through commercial channels.

[0040] Example 1: Construction of MDBK whole genome mutant library and screening of key host factors for BPIV-3 replication

[0041] 1. Recovery and passaging of MDBK cell lines containing Cas9 protein

[0042] First, take out the MDBK cells (MDBK-Cas9) containing Cas9 protein that were previously stored in this laboratory from liquid nitrogen, and quickly put them into a 37°C water bath, shaking continuously during the period to thaw the cells. Put the thawed cells into a sterile operating table, use a pipette to first absorb 5mL of cell culture medium (DMEM containing 5% FBS and 2% double antibody), then absorb the cell suspension, repeatedly blow and beat several times, and inject it into a centrifuge tube. 1000r / min, centrifuge for 5min, and discard the supernatant. Take 2mL of cell culture medium and add it to the centrifuge tube. After repeated blowing and beating, transfer the cells to a six-well plate and culture them in a 37°C, 5% CO2 constant temperature incubator.

[0043] When the cells fill about 95% to 100% of the six-well plate, subculture. The specific operation steps are to take out the six-well plate filled with MDBK-Cas9 cells from the constant temperature incubator, discard the old culture medium, add 1mL PBS to wash, discard PBS, add 1mL trypsin, and incubate in a constant temperature incubator at 37°C and 5% CO2 for 3 minutes. After that, observe whether the cells fall off, add an appropriate amount of cell culture medium and blow repeatedly for more than 10 times, transfer to a 15mL centrifuge tube, 1000r / min, centrifuge for 5 minutes, and discard the supernatant. Add 6mL of cell culture medium, blow and mix, then transfer to 3 wells of the six-well plate, and return to the incubator to continue culturing.

[0044] 2. Lentiviral packaging of sgRNA plasmid library

[0045] Recovery and culture: 293T cells were revived in a 10 cm cell culture dish and passaged after confluence. 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) were taken for lentiviral packaging.

[0046] Transfection: Before transfection, discard the old culture medium, add DMEM culture medium containing 5% FBS and 1% penicillin-streptomycin double antibody and continue to return to the incubator for culture (consider washing with PBS when there are many floating cells); for a 10cm cell culture dish, the total amount of plasmid during lentivirus packaging is 24μg, of which (PMD2.G: PSPAX2: sgRNA plasmid library = 1:2:3); add the corresponding volume of plasmid to 500μL Jetprime Buffer in sequence, gently blow and mix, add 40μL JetprimeRegent, gently blow and mix, let stand at room temperature for 10min, then add to the culture dish, shake gently and return to the incubator; 4-6h after virus packaging, change to 10ml DMEM culture medium containing 5% FBS and 1% double antibody, return to the incubator for continued culture; 24h after the change of medium, add 10ml DMEM culture medium containing 5% FBS and 1% double antibody, mix and return to the incubator.

[0047] Ultracentrifugation: After 60 to 72 hours of virus packaging, observe the cell morphology, collect the cell supernatant (one 50 mL centrifuge tube for every 2 10 cm cell culture dishes), seal with sealing film, centrifuge at 4°C, 3000 rpm / min for 10 min, filter the supernatant with a 0.45 μm filter into an ultracentrifuge tube, and then ultracentrifuge at 30,000 rpm, 4°C for 2.5 hours; then pour out the supernatant, turn it upside down on absorbent paper to absorb the residual liquid, add 120 μL of pre-cooled PBS to each tube to resuspend, blow it open and mix it completely, then transfer the same virus concentrate to the same collection tube and disperse it overnight at 4°C; according to the demand of later experiments, divide it into aliquots and store it at -80°C.

[0048] 3. sgRNA lentiviral library infection and drug screening of library cell lines to obtain MDBK mutant library

[0049] The concentrated lentivirus was used to infect MDBK-Cas9 cells in different volumes (1×10 6 ) to determine the lentiviral titer. Using flow cytometry, 20 μL of lentivirus can produce 1×10 6 The lentiviral infection efficiency of 32.1% was achieved.

[0050] Next, use lentivirus to infect MDBK-Cas9 cells at an MOI of 0.3, requiring a total of 1.4 × 10 8cells can make the sgRNA coverage in the mutant library reach 500×. On the second day after infection, positive cells were selected by puromycin. After 7 days of screening, 1×10 7 The genome of each mutant cell was used as a PCR template for amplification. The high-throughput sequencing primer sequences are shown in Table 1, and the PCR reaction system was prepared according to Table 2. The amplified product was purified, mixed evenly, and then sent for high-throughput sequencing. The sequencing results were analyzed using MAGeCK analysis software. Figure 1 As shown in the figure, the sgRNA coverage reached 92%, the gene coverage reached 99%, and most genes had 4 sgRNAs. This cell mutant library can meet the requirements of full gene screening.

[0051] Table 1 Amplification primers and high-throughput sequencing primer sequence information

[0052]

[0053]

[0054] Table 2 PCR amplification reaction system

[0055]

[0056] 4. Screening of key host factors related to BPIV-3 replication

[0057] First, we explored the optimal infection ratio of BPIV-3 to MDBK-Cas9 cells, diluted the virus in multiples, and infected the cells at infection ratios of 0.5, 0.05, 0.005, and 0.0005, respectively, as a negative control. Observe the cell pathological changes every day. The results showed that when the infection ratio was 0.005, about 50% of the cells died 72 hours after infection. Therefore, we used it as the starting infection ratio for screening.

[0058] Afterwards, we used BPIV-3 to screen the cell mutant library at an MOI of 0.005. After 15 days of screening, many cell colonies could be seen in the culture flask. These cells were collected and expanded, some were frozen and retained in liquid nitrogen, and the other part of the cells continued to be screened, and the process was repeated three more times, but the MOI gradually increased. The genome was extracted from a portion of the surviving cells after the third and fourth rounds of screening, and used as a template for PCR. The high-throughput sequencing primer sequences are shown in Table 1, and the PCR reaction system was prepared according to Table 2. After the PCR products were purified and mixed evenly, they were sent to the company for high-throughput sequencing. The enrichment of sgRNA in the sequencing results was analyzed using MAGeCK software to infer the enrichment of genes.

[0059] 5. Enriched Gene Analysis

[0060] KEGG and GO analyses were performed on the top 50 genes screened in the third and fourth rounds. The results are as follows Figure 2 A. The enriched genes are mainly concentrated in metabolism, positive regulation of IL-6 production, lung development, negative regulation of adipocyte differentiation, etc. The top 100 genes in the two screenings were analyzed, and the results are as follows Figure 2 B, The enriched genes are mainly in the metabolism, exosome, Golgi apparatus and other pathways. After 4 rounds of screening with gradually increasing MOI, the screening results have gradually reached stability.

[0061] Example 2: Construction and verification of SELENON gene-deficient cell line

[0062] 1. Construction of SELENON gene-deficient cell line

[0063] From the sequencing results of the fourth round of screening, the top-ranked SELENON gene was selected to construct a single gene knockout cell line to verify whether the gene is involved in the replication of BPIV-3. The sgRNA targeting the gene was connected to the plasmid vector, and the auxiliary plasmid was used to package the lentivirus and infect MDBK-Cas9 cells. The sgRNA sequence and plasmid construction primer sequence information are shown in Table 3. The single clone was selected by the limiting dilution method, and the cell genome was extracted after expansion culture. PCR amplification was performed using this as a template. The PCR amplification primer sequence is shown in Table 4, and finally the genotype was determined by sequencing. The results are shown in Figure 3 , and found that the SELENON gene had lost some bases (not a multiple of 3).

[0064] Table 3 sgRNA sequence and plasmid construction primer sequence information

[0065]

[0066] Table 4 Primer sequence information for amplifying target fragments

[0067]

[0068] 2. Transcriptional verification of SELENON gene-deficient cell lines

[0069] The Trizol method was used to extract RNA from wild-type MDBK and the gene knockout monoclonal cell line numbered SELENON-KO, and the RNA was reverse transcribed into cDNA using a reverse transcription kit (Company: Nanjing Novezan Biotechnology Co., Ltd.; Catalog No.: R223-01), and relative quantitative qPCR was performed to verify the expression of candidate genes at the transcription level. The primer sequences for relative quantitative qPCR are shown in Table 5, and the relative quantitative qPCR reaction system is prepared according to Table 6. The program is 95℃ pre-denaturation for 5min, the number of cycles is 1; 95℃ denaturation for 10s, 60℃ annealing for 30s, and the instrument defaults to the melting curve. The results are shown in Figure 4 Compared with wild-type MDBK cells, the gene knockout monoclonal cell line numbered SELENON-KO showed a significant decrease in gene expression at the transcriptional level.

[0070] Table 5 Relative quantitative qPCR primer sequence information

[0071]

[0072] Table 6 Rt-qPCR amplification reaction system

[0073]

[0074]

[0075] 3. Verification of EdU cell proliferation level in SELENON gene-deficient cells

[0076] To evaluate the cell proliferation level after knockout of candidate genes, wild-type MDBK and SELENON-KO knockout cell lines were cultured at 5 × 10 5 Cells were seeded into 6-well plates. The cells were transferred into DMEM medium supplemented with 10% FBS and 1% double antibody and incubated in an incubator at 37°C and 5% CO2. After incubation for 24 hours, BeyoClick with Alexa Fluor 555 (Company: Beyotech; Product No.: C0075s) was used to TM EdU cell proliferation assay was performed using the EdU cell proliferation kit, and cell nuclei were stained with DAPI (company: Bio-Tech; catalog number: C1005) for 10 min at room temperature and 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 5 The proliferation ability of SELENON gene knockout cells was not significantly changed compared with wild-type MDBK cells, and this cell line can be used for subsequent verification experiments.

[0077] Example 3: Using TCID 50Detection of the anti-BPIV-3 ability of SELENON gene-deficient MDBK monoclonal cells by absolute quantitative qPCR

[0078] 1. TCID of SELENON gene-deficient cells against BPIV-3 virus 50 verify

[0079] Wild-type MDBK and SELENON gene-deficient cell lines were revived in six-well plates, and when the cells grew to 90%, they were inoculated with BPIV-3 at an MOI of 0.1. Virus samples were collected 12, 18, 24, 30, and 36 hours after inoculation, and the TCID of BPIV-3 in the wild-type MDBK and SELENON gene-deficient cell line samples was detected. 50 , and observe their phenotypic differences. Figure 6 After infection with BPIV-3, the virus titer of the SELENON gene-deficient cell line was significantly reduced compared with that of the wild-type MDBK.

[0080] 2. Absolute quantitative qPCR validation of SELENON gene-deficient cells against BPIV-3 virus

[0081] The virus sample obtained after 24 hours of infection with BPIV-3 was used to extract viral RNA using a virus RNA extraction kit (Company: Nanjing Novezan Biotechnology Co., Ltd.; Catalog No.: RM402-01), and the RNA was reverse transcribed into cDNA using a reverse transcription kit (Company: Nanjing Novezan Biotechnology Co., Ltd.; Catalog No.: R223-01), and the cDNA product was used as a template for absolute quantitative qPCR. The primer sequences for absolute quantitative qPCR are shown in Table 7, and the absolute quantitative qPCR reaction system was prepared according to Table 6 above. The amplification program is: pre-denaturation at 95°C for 5 minutes, the number of cycles is 1; denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and the instrument defaults to the melting curve. The results are as follows Figure 7 , same as TCID 50 The results were consistent, and compared with wild-type MDBK, the number of BPIV-3 virus copies amplified by the SELENON gene-deficient cell line was significantly reduced. This result proves that the SELENON gene is a host factor involved in BPIV-3 replication.

[0082] Table 7 Absolute quantitative qPCR primer sequence information

[0083]

[0084] Example 4: SELENON gene complementation verification

[0085] 1. Construction of SELENON gene complementation plasmid

[0086] RNA was extracted from wild-type MDBK cells, and cDNA was obtained by reverse transcription. The SELENON gene was amplified using this cDNA as a template. A Flag tag was added before the stop codon at the end of the target sequence. The amino acid near the NGG position of the sgRNA was mutated to a synonymous amino acid to obtain the SELENON gene complementation sequence. The pLVX-EGFP-IRES-Neo vector was double-digested with EcoRI (Company: New England Biolabs; Catalog No.: R0101V) and BamHI (Company: New England Biolabs; Catalog No.: R0136V), and the 8269bp vector band was recovered. The target band and the vector band were connected using homologous recombinase (Company: Nanjing Novezan Biotechnology Co., Ltd.; Catalog No.: C112-01) to construct the pLVX-c SELENON-flag complementation plasmid. The connection was carried out overnight at 4℃, and the bacteria were picked for identification and sequenced to obtain the pLVX-c SELENON-flag complementation plasmid. The endotoxin-free plasmid was extracted using a plasmid mini-extraction kit (company: Omega Bio-Tek; catalog number: D6950-02) and stored at -80°C. The restriction enzyme reaction system of the pLVX-EGFP-IRES-Neo vector is shown in Table 8, the complemented sequence fragment and vector restriction enzyme ligation system is shown in Table 9, and the homologous recombination amplification primer sequence is shown in Table 10.

[0087] Table 8 Enzyme Digestion Reaction System

[0088]

[0089] Table 9 Recombination system of complemented sequence fragments and vector restriction enzyme digestion

[0090]

[0091]

[0092] Table 10 Homologous recombination amplification primer sequence information

[0093]

[0094] 2. Obtaining SELENON gene complemented cell lines

[0095] Refer to the method of step 2 of Example 1 to carry out lentiviral packaging of pLVX-c SELENON-flag complementing plasmid, that is, replace the "sgRNA plasmid library" in the step with the pLVX-c SELENON-flag complementing plasmid in the above step.

[0096] The SELENON-KO cell line was revived in a six-well plate, and after it was full, it was subcultured at a ratio of 1:3. When the cells were full to about 60%, the old culture medium was discarded, and 1 mL of cell maintenance solution (DMEM containing 2% FBS + 1% double antibody) was added to each well, followed by 1 μL of polybreme, and then 40 μL of SELENON complement plasmid lentivirus was added and mixed. After incubation 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, 2 mL of DMEM culture medium containing 5% FBS, 1% double antibody and 400 μg / mL neomycin was replaced for drug screening. After all the negative control drug screening wells died, the positive control and negative control were subcultured (Note: the positive control wells were subcultured in a six-well plate at a ratio of 1:2, and the negative control was subcultured in two wells, one of which was used as the negative control drug screening group). In this way, the drug screening effect lasted for a total of 7 days to obtain the MDBK cell line with SELENON gene complementation. The total RNA of the complemented cells was extracted using Trizol, and the relative quantitative method was used to verify whether the complementation experiment was successful. The results are as follows: Figure 8 , SELENON gene was successfully restored.

[0097] 3. Phenotypic Identification of SELENON Gene-complemented Cell Lines

[0098] Referring to Example 3, wild-type MDBK cells, SELENON gene knockout cell lines, and SELENON gene complemented cells were infected with BPIV-3 virus, and TCID 50 To verify whether the phenotype has been restored, the results are as follows Fig. 9 After SELENON gene complementation, susceptibility to BPIV-3 is restored.

Claims

1. Application of SELENON protein or a gene encoding the protein in regulating host antiviral activity, wherein the virus is bovine parainfluenza virus type 3 (BPIV-3).

2. The use according to claim 1, characterized in that: Knocking out or silencing the SELENON gene can improve the host's resistance to infection and inhibit the proliferation of BPIV-3, while complementing the SELENON gene makes the host susceptible to BPIV-3.

3. Use of an agent for knocking out or silencing the SELENON gene in the preparation of anti-BPIV-3 drugs and animal cells or species.

4. An anti-BPIV-3 drug comprising an agent for knocking out or silencing the SELENON gene.

5. The anti-BPIV-3 drug according to claim 4, characterized in that: The reagent includes a Cas9 gene editing protein or an expression vector thereof, and a gRNA or an expression vector thereof that guides the Cas9 gene editing protein to specifically bind to the SELENON gene.

6. A BPIV-3 resistant bovine kidney cell line, wherein the SELENON gene of the bovine kidney cell line is knocked out or silenced, and the expression of the endogenous SELENON gene is absent or reduced, thereby having the ability to resist BPIV-3.

7. A method for preparing an anti-BPIV-3 bovine kidney cell line, characterized in that The following steps are involved: (1) Design sgRNA sequences and primers targeting the SELENON gene and construct sgRNA expression plasmids; (2) Lentiviral vector packaging of sgRNA expression plasmid; (3) using a lentiviral vector containing an sgRNA expression plasmid to infect bovine kidney cells containing the Cas9 protein, and obtaining a polyclonal bovine kidney cell line in which the SELENON gene was knocked out; (4) Monoclonal cells were selected from the polyclonal bovine kidney cell line in which the SELENON gene was knocked out.

8. The method for preparing the anti-BPIV-3 bovine kidney cell line according to claim 7, characterized in that: The amino acid sequence encoded by the SELENON gene is shown in SEQ ID NO.

1.

9. The method for preparing the anti-BPIV-3 bovine kidney cell line according to claim 8, characterized in that: The sgRNA sequence is shown in SEQ ID NO.

2.

10. The method for preparing the anti-BPIV-3 bovine kidney cell line according to claim 9, characterized in that: The primer sequences are shown in SEQ ID NO.3 and 4.

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