Application of the SELENON gene or its encoded protein in regulating host resistance to BPIV-3

Abstract

This invention discloses the application of the SELENON gene in regulating host resistance to Bovine parainfluenza virus type 3 (BPIV-3). Using CRISPR / Cas9 gene editing technology, a SELENON gene knockout MDBK cell line and a cell model resistant to BPIV-3 infection were constructed. The results showed that the gene knockout cells exhibited significant resistance to BPIV-3 infection, and the proliferation rate of BPIV-3 in gene knockout cells was significantly lower than in wild-type cells. Reintroduction of the SELENON gene resulted in the loss of antiviral activity in the cells. These results indicate that the SELENON gene plays an important role in regulating host antiviral activity. This invention identifies the antiviral regulatory function of the SELENON gene at the cellular level and, for the first time, discovers that this gene can serve as a potential target for gene-edited cells and animal design, applicable to disease-resistant breeding and the development of drugs for the prevention and treatment of bovine BPIV-3 infection. This has significant implications for reducing drug abuse and preventing disease outbreaks.

Description

Technical Field

[0001] This invention belongs to the field of animal infectious disease prevention and control technology, and relates to the application of the SELENON gene in regulating host resistance to Bovine parainfluenza virus type 3 (BPIV-3). Background Technology

[0002] Bovine parainfluenza virus type 3 (BPIV-3) is an important pathogen causing respiratory diseases in cattle. Belonging to the family Paramyxoviridae and the genus Respirovirus, it is an enveloped, unsegmented, single-stranded negative-sense RNA virus, approximately 15,000 bp in length. Bovine parainfluenza caused by BPIV-3 is a contagious, febrile, and acute respiratory disease in cattle, characterized by fever, cough, general weakness, lethargy, and often respiratory distress. It can also cause abortion or reproductive problems in cows. Severe cases can lead to death. This pathogen often causes mixed infections with bovine respiratory syncytial virus (BRSV), bovine viral diarrhea / Mucosaldisease (BVDV), infectious bovine rhinotracheitis virus (IBRV), Pasteurella, Mansonia, and Mycoplasma, a condition known as Bovine respiratory disease complex (BRDC), commonly called "transport fever." The main symptoms in affected cattle include high fever, purulent conjunctivitis, lacrimation, nasal discharge, frequent coughing, difficulty breathing, and frothy saliva. Currently, my country lacks effective drugs and vaccines to combat BPIV-3; therefore, developing more effective vaccines and drugs for prevention and treatment is crucial.

[0003] Previous studies have mostly used RNA interference (RNAi) libraries for genetic screening, but this method has drawbacks such as low inhibition efficiency and high off-target rate. CRISPR / Cas9 technology, as a third-generation gene editing technology, has the advantages of being economical, simple to operate, accurate, and efficient, and has been successfully applied in whole-genome screening of various mammals, including pigs, humans, mice, and African green monkeys.

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

[0005] The first objective of this invention is to screen host factors involved in BPIV-3 replication.

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

[0007] First, the MDBK cell line containing Cas9 protein, previously selected by the Ruminant Pathogen Division of the State Key Laboratory of Agricultural Microbiology at Huazhong Agricultural University, was revived and cultured. Lentiviral viruses were packaged from an sgRNA plasmid library constructed earlier in the laboratory. MDBK-Cas9 cells were then infected with these lentiviruses. After puromycin screening, high-throughput sequencing of the mutant cell library revealed a gene coverage of 99.62% and an sgRNA coverage of 92.12%, meeting the screening requirements. Next, the mutant cell library was infected with BPIV-3 and screened four times. Surviving infected cells were collected and subjected to high-throughput sequencing to identify host factors that affect BPIV-3 replication. Finally, single-gene deletion cell lines were constructed from these candidate genes and infected with BPIV-3. Absolute quantification of the BPIV-3 genome copy number revealed that deletion of each candidate gene significantly inhibited BPIV-3 replication, while gene restoration resulted in the loss of antiviral activity.

[0008] The above results demonstrate that a whole-genome mutant library can reveal a number of unknown key host factors involved in BPIV-3 replication, thus 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 whole-genome mutant library also has the following advantages:

[0009] 1) The designed sgRNA library contains 23,265 protein-coding genes, covering almost all fully annotated protein-coding genes in the bovine genome, providing broad coverage. Furthermore, four sgRNAs were designed for each gene, targeting the upstream position of the gene's open reading frame, ensuring gene knockout efficiency. The maximum allowable number of mismatched bases for each sgRNA is 5 bp, reducing the risk of off-target effects.

[0010] 2) This 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 using it. The sgRNA plasmid library can be passaged multiple times, and the constructed cell mutant library can also be passaged to a limited extent, ensuring that the sgRNA content and coverage will not change significantly.

[0011] A second objective of this invention is to provide the SELENON gene, screened using the above method, for regulating host resistance. Functions related to BPIV-3.

[0012] To achieve the above objectives, this invention utilizes CRISPR / Cas9 gene editing technology to construct a targeting vector for the SELENON gene in MDBK cells. Following vector transfection and cell screening, SELENON gene knockout cell lines and a cell model resistant to BPIV-3 viral infection were established. Virological testing revealed that SELENON gene knockout cells exhibited significant resistance to BPIV-3 infection, and the viral proliferation rate in knockout cells was significantly lower than in wild-type cells. This indicates that at the cellular level, SELENON gene knockout effectively inhibits viral infection and proliferation. Conversely, reintroduction of the SELENON gene resulted in the loss of antiviral activity, demonstrating the crucial role of the SELENON gene in BPIV-3 replication and the regulation of host antiviral capabilities. This invention utilizes a CRISPR / Cas9-mediated gene knockout method to establish a cell model effectively resistant to BPIV-3 infection, confirming the role of the SELENON gene as a target in antiviral infection. This strategy can be applied to the preparation of SELENON gene-modified BPIV-3-resistant cells and animals, as well as the development of novel drugs.

[0013] A third objective of this invention is to provide reagents for knocking out or silencing the SELENON gene in the preparation of antibodies. Uses of BPIV-3 in drugs and in animal cells and varieties.

[0014] To achieve the above objectives, this invention utilizes CRISPR / Cas9 gene editing technology to knock out the SELENON gene in MDBK cells. It was found that the gene-knockout cells exhibited significant resistance to BPIV-3 infection, with a significantly reduced viral titer. Therefore, the CRISPR / Cas9 reagent used to knock out the SELENON gene can be used to prepare anti-BPIV-3 drugs and to prepare animal cells and strains resistant to BPIV-3 infection. Further, the CRISPR / Cas9 reagent comprises: a Cas9 gene-editing protein or its expression vector, and an sgRNA or its expression vector that guides the Cas9 gene-editing protein to specifically bind to the SELENON gene. However, this invention is not limited to reagents developed based on CRISPR / Cas9 technology; other reagents designed using other gene knockout or silencing methods well-known in the art to knock out or silence the target SELENON gene are also within the scope of protection of this invention.

[0015] A fourth objective of this invention is to provide a drug for treating BPIV-3.

[0016] This drug contains a reagent for knocking out or silencing the SELENON gene. Further, 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, but is not limited thereto. This invention has verified at the cellular level that this drug can effectively knock out or silence the SELENON 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, especially cattle, to achieve the same function.

[0017] The fifth objective of this invention is to provide a bovine kidney cell line in which the SELENON gene is knocked out or silenced. These bovine kidney cells possess anti-BPIV-3 capabilities and show promise for applications in virus isolation and identification, as well as research on antiviral host factors.

[0018] The sixth objective of this invention is to provide a bovine kidney cell line with the SELENON gene knocked out or silenced. Preparation method. The method includes the following steps:

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

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

[0021] (3) Bovine kidney cells containing Cas9 protein were infected with a lentiviral vector containing a sgRNA expression plasmid to obtain a polyclonal bovine kidney cell line with the SELENON gene knocked out.

[0022] (4) Select monoclonal cells from polyclonal bovine kidney cell lines.

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

[0024] This invention successfully constructed SELENON gene knockout bovine kidney cells using the CRISPR / Cas9 gene editing method. Experimental results show that these cells can resist BPIV-3 infection, inhibit viral titer and expression level, and the method has high reproducibility.

[0025] The present invention has the following advantages:

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

[0027] 2) The SELENON gene is expected to serve as a target for drug design in the screening of candidate compounds for anti-BEV infection. Test compounds are added to cell models 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) This 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 highly efficient, 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. Attached Figure Description

[0030] Figure 1 Sequencing statistical analysis of the MDBK cell mutant library. A. MAGeCK analysis of sequencing results of the cell mutant library; B. Number of sgRNAs for each gene in the cell mutant library.

[0031] Figure 2 Enrichment 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: Results of the third round of screening of the cell mutant library using BPIV-3; BPIV-3_4th: Results of the fourth round of screening of the cell mutant library using BPIV-3.

[0032] Figure 3 Genotype of a single deletion clone cell line of the SELENON gene. WT: Wild-type MDBK; "-" represents a missing base.

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

[0034] Figure 5 Validation 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 in SELENON gene-deficient cells resisting 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-deleted cells' resistance to the virus. WT: Wild-type MDBK, ***p<0.001, mean±SEM (n=3).

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

[0038] Figure 9 SELENON gene complementation to expand BPIV-3 TCID 50 Phenotypic validation. WT: Wild-type MDBK, ns: No significant difference, **p<0.01, ***p<0.001, mean±SEM (n=3). Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of protection of the present invention. Various modifications or equivalent substitutions made by those skilled in the art based on the following embodiments should also be considered to fall within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out according to conventional conditions or reference books such as "Molecular Cloning: A Laboratory Manual" (4th edition), or according to the methods recommended in the manufacturer's operating manual. Materials whose sources are not specified in the embodiments, such as MDBK cells, 293T cells, packaging plasmids, T vectors, etc., are all commonly used materials well known in the art, which can be constructed by oneself according to literature reports or obtained through commercial means.

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

[0041] 1. Resuscitation and passage of MDBK cell lines containing Cas9 protein

[0042] First, remove the previously preserved MDBK cells containing Cas9 protein (MDBK-Cas9) from liquid nitrogen and quickly place them in a 37°C water bath, shaking constantly to thaw the cells. Place the thawed cells on a sterile work surface and pipette 5 mL of cell culture medium (DMEM containing 5% FBS and 2% penicillin-dextrose antibodies), then pipette the cell suspension, repeatedly pipetting several times, and transfer to a centrifuge tube. Centrifuge at 1000 rpm for 5 min, discarding the supernatant. Add 2 mL of cell culture medium to the centrifuge tube, repeatedly pipetting to ensure homogeneity, and then transfer the cells to a six-well plate. Incubate at 37°C in a 5% CO2 incubator.

[0043] When the cells have confluently covered approximately 95%–100% of the six-well plate, proceed with passage culture. The specific steps are as follows: Remove the six-well plate filled with MDBK-Cas9 cells from the incubator, discard the old culture medium, add 1 mL of PBS for washing, discard the PBS, add 1 mL of trypsin, and incubate at 37°C with 5% CO2 for 3 minutes. Observe whether the cells have detached. Add an appropriate amount of cell culture medium and repeatedly pipette the plate about 10 times. Transfer the plate to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add 6 mL of cell culture medium, mix well, and transfer the mixture to three wells of the six-well plate. Return the plate to the incubator for further culture.

[0044] 2. Lentiviral packaging sgRNA plasmid library

[0045] Resuscitation and culture: Resuscitate 293T cells in 10cm cell culture dishes, passage them after they reach confluence, and take 293T cells in good growth condition from about 3 passages (10cm cell culture dish, 90%-95% confluence, fresh antibiotic-free DMEM medium containing 10% FBS) for lentiviral packaging.

[0046] Transfection: Before transfection, discard the old culture medium and add DMEM medium containing 5% FBS and 1% penicillin-streptomycin antibiotics. Return the culture dish to the incubator (if there are many floating cells, consider washing with PBS once). For a 10cm cell culture dish, the total plasmid content 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, gently pipette to mix, then add 40μL JetprimeRegent, gently pipette to mix, let stand at room temperature for 10min, then add to the culture dish, gently shake, and return to the incubator. 4-6 hours after virus packaging, replace with 10ml of DMEM medium containing 5% FBS and 1% penicillin-streptomycin antibiotics, and return to the incubator for further culture. 24 hours after the medium change, add another 10ml of DMEM medium containing 5% FBS and 1% penicillin-streptomycin antibiotics, mix well, and return to the incubator.

[0047] Ultracentrifugation: After 60–72 hours of virus packaging, observe cell morphology and collect cell supernatant (two 10cm cell culture dishes per 50mL centrifuge tube). Seal the tubes with sealing film and centrifuge at 3000rpm / min for 10 minutes at 4°C. Filter the supernatant through a 0.45μm filter into an ultracentrifuge tube and centrifuge at 30000rpm at 4°C for 2.5 hours. Afterward, discard the supernatant, invert the tubes onto absorbent paper to remove any remaining liquid, add 120μL of pre-chilled PBS to each tube for resuspending, thoroughly mix, and transfer the same concentrated virus solution to the same collection tube for diffusion overnight at 4°C. Aliquot according to the required amount for later experiments and store at -80°C.

[0048] 3. Obtaining the MDBK mutant library by infecting the constructed cell lines with the sgRNA lentiviral library and drug screening.

[0049] Concentrated lentiviruses were used to infect MDBK-Cas9 cells at different volumes (1×10⁻⁶). 6 Lentiviral titers were determined. Flow cytometry was used to detect that 20 μL of lentivirus could produce 1 × 10⁻⁶ lentivirus titers. 6 The lentiviral infection efficiency reached 32.1% per cell.

[0050] Next, MDBK-Cas9 cells were infected with lentivirus at an MOI of 0.3, requiring a total of 1.4 × 10⁻⁶ cells. 8One cell can achieve 500× sgRNA coverage in the mutant library. Positive cells were screened by puromycin on day 2 post-infection. Seven days after screening, 1×10⁻⁶ cells were extracted using the TIANamp Genomic DNA Kit (company: Tiangen, catalog number: DP304-02). 7 The genome of a mutant cell was used as a PCR template for amplification. High-throughput sequencing primer sequences are shown in Table 1, and the PCR reaction system was prepared according to Table 2. The amplification products were purified, thoroughly mixed, and then sent for high-throughput sequencing. The sequencing results were analyzed using MAGeCK analysis software, and the results are as follows: Figure 1 As shown, the sgRNA coverage reached 92%, and the gene coverage reached 99%, with the vast majority of genes having four sgRNAs. This cell mutant library can meet the requirements for whole-genome screening.

[0051] Table 1. Sequence information of amplification primers and high-throughput sequencing primers.

[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 determined the optimal infection ratio of BPIV-3 to MDBK-Cas9 cells. The virus was serially diluted and used to infect cells at infection ratios of 0.5, 0.05, 0.005, and 0.0005, with a negative control included. Cytopathic effects were observed daily. The results showed that at an infection ratio of 0.005, approximately 50% of the cells died 72 hours post-infection. Therefore, we used this as the initial infection ratio for screening.

[0058] Next, we used BPIV-3 to screen the cell mutant library at an MOI of 0.005. After 15 days of screening, many cell colonies were observed in the culture flasks. These cells were collected and cultured on a large scale. A portion was cryopreserved in liquid nitrogen, while the other portion was screened again, repeated three times with the MOI gradually increasing. Genomic DNA 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 purifying and thoroughly mixing the PCR products, 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 gene enrichment.

[0059] 5. Enrichment gene analysis

[0060] KEGG and GO analyses were performed on the top 50 genes selected in the third and fourth rounds of screening, and the results are as follows: Figure 2 A. The enriched genes are mainly concentrated in metabolism, positive regulation of IL-6 production, lung development, and negative regulation of adipocyte differentiation; analysis of the top 100 genes from both screenings yielded the following results: Figure 2 B. The enriched genes are mainly found in metabolic, exosome, and Golgi pathways. After four rounds of screening with gradually increasing MOI, the screening results have gradually stabilized.

[0061] Example 2: Construction and validation of SELENON gene deletion cell lines

[0062] 1. Construction of SELENON gene deletion cell lines

[0063] The top-ranked SELENON gene was selected from the sequencing results of the fourth round of screening to construct a single-gene knockout cell line to verify whether this gene is involved in BPIV-3 replication. The sgRNA of the target gene was ligated into a plasmid vector, and lentivirus was packaged using a helper plasmid to infect MDBK-Cas9 cells. The sgRNA sequence and primer sequences for plasmid construction are shown in Table 3. Single clones were selected using the limiting dilution method, and after amplification culture, the cell genome was extracted. This genome was used as a template for PCR amplification. The PCR primer sequences are shown in Table 4. Finally, sequencing was used to determine the genotype. The results are as follows: Figure 3 It was discovered that the SELENON gene has lost some bases (not multiples of 3).

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

[0065]

[0066] Table 4 Primer sequence information for amplifying the target fragment

[0067]

[0068] 2. Validation of transcriptional levels in SELENON gene-deleted cell lines

[0069] RNA was extracted from wild-type MDBK and gene knockout monoclonal cell lines (SELENON-KO) using the Trizol method. The RNA was then reverse transcribed into cDNA using a reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd.; catalog number: R223-01). Relative quantitative qPCR was then performed to verify the transcriptional expression of candidate genes. Primer sequences for relative quantitative qPCR are shown in Table 5, and the reaction system was prepared according to Table 6. The program was: 95℃ pre-denaturation for 5 min, cycle number 1; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, with the instrument's default melting curve. Results are shown below. 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. Primer sequence information for relative quantitative qPCR

[0071]

[0072] Table 6. Rt-qPCR amplification reaction system

[0073]

[0074]

[0075] 3. Verification of EDU cell proliferation level in SELENON gene-deleted cells

[0076] To assess cell proliferation levels after knocking out candidate genes, wild-type MDBK and knockout cell lines (SELENON-KO) were cultured at 5 × 10⁻⁶ cells per cell line. 5 Cells were seeded into 6-well plates. Cells were then transferred to DMEM medium supplemented with 10% FBS and 1% penicillin antibiotics and incubated at 37°C and 5% CO2. After 24 hours of incubation, cells were seeded using BeyoClick microsodium fluorocarbonate (BioYunTian; catalog number: C0075s) containing Alexa Fluor555 (BioYunTian; catalog number: C0075s). TM EdU cell proliferation was measured using an EdU cell proliferation assay kit, and cell nuclei were stained with DAPI (Beyotime; catalog number: C1005) for 10 min at room temperature and in the dark. Stained cells were observed using a fluorescence microscope. The percentage of EdU-positive cells was calculated using ImageJ software. Imaging was performed on three independent wells, with one field of view randomly captured from each well. Results are shown below. Figure 5 Compared with wild-type MDBK cells, the proliferation capacity of SELENON gene knockout cells did not change significantly, and this cell line can be used for subsequent validation experiments.

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

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

[0079] Wild-type MDBK and SELENON gene-deleted cell lines were revived in six-well plates. When the cells reached 90% confluence, BPIV-3 was inoculated at an MOI of 0.1. Virus samples were collected at 12, 18, 24, 30, and 36 hours after inoculation, and the TCID of BPIV-3 in the wild-type MDBK and SELENON gene-deleted cell line samples was detected. 50 phenotypic differences were observed. The results were as follows: Figure 6 After infection with BPIV-3, the viral titer of the SELENON gene-deleted cell line was significantly reduced compared with that of wild-type MDBK.

[0080] 2. Absolute quantitative qPCR verification of SELENON gene-deleted cells' resistance to BPIV-3 virus

[0081] Viral RNA was extracted from virus samples obtained 24 hours after BPIV-3 infection using a viral RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.; catalog number: RM402-01). The RNA was then reverse transcribed into cDNA using a reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd.; catalog number: R223-01). The cDNA product was used as a template for absolute quantitative qPCR. 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: 95℃ pre-denaturation for 5 min, cycle number 1; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, with the instrument's default melting curve. Results are as follows: Figure 7 Same as TCID 50 The results were consistent; compared to wild-type MDBK, the BPIV-3 viral copy number expanded in the SELENON gene-deleted cell line was significantly reduced. This result demonstrates that the SELENON gene is a host factor involved in BPIV-3 replication.

[0082] Table 7 Primer sequence information for absolute quantitative qPCR

[0083]

[0084] Example 4: Verification of SELENON gene complementation

[0085] 1. Construction of SELENON gene complementation plasmid

[0086] RNA was extracted from wild-type MDBK cells, and cDNA was obtained through reverse transcription. The SELENON gene was then amplified using cDNA as a template, with a Flag tag added before the stop codon at the end of the target sequence. Amino acids near the NGG position of the sgRNA were mutated to synonymous amino acids to obtain the SELENON gene complement sequence. The pLVX-EGFP-IRES-Neo vector was digested with EcoRI (New England Biolabs; catalog number: R0101V) and BamHI (New England Biolabs; catalog number: R0136V), and the 8269bp vector band was recovered. The target band and the vector band were ligated using a homologous recombinase (Nanjing Novizan Biotechnology Co., Ltd.; catalog number: C112-01) to construct the pLVX-c SELENON-flag complement plasmid. Ligation was performed overnight at 4℃, followed by bacterial identification and sequencing to obtain the pLVX-c SELENON-flag complement plasmid. The endotoxin-free plasmid was extracted using a plasmid miniprep kit (Omega Bio-Tek; catalog number: D6950-02) and stored at -80°C. The enzyme digestion system of the pLVX-EGFP-IRES-Neo vector is shown in Table 8, the enzyme digestion and ligation system of the complemented sequence fragment and the vector is shown in Table 9, and the primer sequences for homologous recombination amplification are shown in Table 10.

[0087] Table 8 Enzyme digestion reaction system

[0088]

[0089] Table 9. Enzyme digestion and ligation system of the supplemented sequence fragment and vector.

[0090]

[0091]

[0092] Table 10 Primer sequence information for homologous recombination amplification

[0093]

[0094] 2. Obtaining the SELENON gene complementation cell line

[0095] Refer to step 2 of Example 1 to package the pLVX-c SELENON-flag complement plasmid for lentivirus packaging, that is, replace the "sgRNA plasmid library" in the step with the pLVX-c SELENON-flag complement plasmid in the above step.

[0096] SELENON-KO cell lines were revived in six-well plates and passaged 1:3 after confluence. When the cells reached approximately 60% confluence, the old culture medium was discarded, and 1 mL of cell maintenance medium (DMEM containing 2% FBS and 1% penicillin antibody) was added to each well. Then, 1 μL of polybreme was added, followed by 40 μL of SELENON complement plasmid lentivirus. After incubation for 24 h, the old culture medium was discarded, and 2 mL of culture medium containing 5% FBS and 1% penicillin antibody was added. After another 24 h of incubation, the medium was replaced with 2 mL of DMEM containing 5% FBS, 1% penicillin antibody, and 400 μg / mL neomycin for drug screening. After all cells in the negative control screening wells died, the positive and negative controls were passaged (note: positive control wells were passaged 1:2 in six-well plates, and negative control wells were passaged in two wells, one for the negative control drug screening group). This drug screening process was repeated for a total of 7 days to obtain the SELENON gene complemented MDBK cell line. Total RNA was extracted from the replaced cells using Trizol, and a relative quantification method was used to verify the success of the replacement experiment. The results are as follows: Figure 8 The SELENON gene was successfully reintroduced.

[0097] 3. Phenotypic identification of SELENON gene complementation cell lines

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

Claims

1. The use of a reagent for knocking out or silencing the bovine SELENON gene in the preparation of a bovine anti-BPIV-3 drug, wherein the protein sequence encoded by the bovine SELENON gene is shown in SEQ ID NO.

1.

2. The application of a reagent for knocking out or silencing the bovine SELENON gene in the preparation of a bovine kidney cell line resistant to BPIV-3, wherein the application is to induce or reduce the expression of the endogenous bovine SELENON gene, thereby achieving anti-BPIV-3 activity, the protein sequence encoded by the bovine SELENON gene being shown in SEQ ID NO.

1.

3. A method for preparing an anti-BPIV-3 bovine kidney cell line, characterized in that... Includes the following steps: (1) Design the sgRNA sequence of the bovine SELENON gene and primers for amplifying the sgRNA sequence, and construct an sgRNA expression plasmid. The protein sequence encoded by the bovine SELENON gene is shown in SEQ ID NO.1, and the sgRNA sequence is shown in SEQ ID NO.

2. (2) Lentiviral vector packaging of sgRNA expression plasmid; (3) Bovine kidney cells containing Cas9 protein were infected with lentiviral vectors containing sgRNA expression plasmids to obtain polyclonal bovine kidney cell lines with bovine SELENON gene knockout. (4) Select monoclonal cells from a polyclonal bovine kidney cell line in which the bovine SELENON gene has been knocked out.

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

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