Use of WNT5A gene or its encoded protein in regulating host resistance to BPIV-3

Abstract

The application discloses application of WNT5A gene in regulation and control of host resistance to bovine parainfluenza virus type 3 (BPIV-3). By using CRISPR / Cas9 gene editing technology, a WNT5A gene knockout MDBK cell line and a BPIV-3 infection resistant cell model are constructed, it is found that the gene knockout cell shows significant resistance to BPIV-3 infection, the proliferation speed of BPIV-3 on the gene knockout cell is significantly lower than that of the wild type cell, and the complementation of the WNT5A gene leads to the disappearance of the antiviral effect of the cell. The above results show that the WNT5A gene plays an important role in regulation and control of host resistance to virus. The application identifies the antiviral regulation function of the WNT5A gene at the cell level, for the first time finds that the gene can be used in resistance breeding and research and development of prevention and treatment drugs for bovine BPIV-3 infection as a potential target point for gene editing cell and animal design, and has great significance for reducing drug abuse and avoiding epidemic occurrence.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal infectious disease prevention and treatment, and relates to application of WNT5A gene in regulation and control of host resistance to bovine parainfluenza virus type 3 (BPIV-3). BACKGROUND

[0002] Bovine parainfluenza virus type 3 (BPIV-3) is an important pathogen that can cause bovine respiratory disease, belongs to the paramyxoviridae family, and is a kind of single-stranded negative-strand RNA virus with a capsule and no segment, with a total length of about 15000 bp. BPIV-3 causes bovine parainfluenza, which is a contact, heat, acute bovine respiratory infectious disease, with fever, cough, general weakness, and depression as the main symptoms, and also often shows respiratory distress, abortion or reproductive disorders in cows. Severe cases can lead to death. The pathogen often mixes with bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), Pasteurella multocida, Mannheimia, mycoplasma and other pathogens to cause mixed infection, that is, 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 cough, and difficulty breathing, and foamy sputum. At present, China 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] Previous studies mostly used the method of RNA interference (RNAi) library for genetic screening, but this method has the disadvantages of low inhibition efficiency and high off-target rate. As the third generation of gene editing technology, CRISPR / Cas9 technology has the advantages of economy, simple operation, accuracy and high efficiency, and has been successfully applied in whole genome screening of pigs, humans, mice, African green monkeys and other mammals.

[0004] Based on this, the application uses CRISPR / Cas9 technology to establish a bovine whole genome cell mutant library, and uses it to screen bovine parainfluenza virus 3 susceptible genes, to provide technical support for further developing vaccines and drugs that can more effectively resist BPIV-3. Through bovine whole genome screening, the application finds that the Wnt family member 5A (Wnt family member 5A, WNT5A) gene plays an important role in regulating host resistance to BPIV-3 infection. SUMMARY

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

[0006] To achieve the above-mentioned purpose, the applicant uses CRISPR / Cas9 technology to construct a bovine kidney cell (Madin-Darby bovine kidney, MDBK) whole genome mutant library, infects the mutant library with BPIV-3, and then performs high-throughput sequencing to find enriched genes and performs verification. The specific implementation scheme is as follows:

[0007] Firstly, the MDBK cell monoclonal cell line containing Cas9 protein selected by the author's laboratory in the early stage was resuscitated and cultured, the sgRNA plasmid library constructed by the laboratory in the early stage was packaged into lentivirus, the MDBK-Cas9 cells were infected with the lentivirus packaged with the sgRNA plasmid library, and after puromycin screening, high-throughput sequencing was performed on the cell mutant library, and it was found that the coverage of the genes reached 99.62%, and the coverage of the sgRNA reached 92.12%, meeting the requirements of screening. Then, the cell mutant library was infected with BPIV-3 and subjected to 4 rounds of screening, the surviving cells after infection were collected, high-throughput sequencing was performed, and host factors that can affect BPIV-3 replication were screened. Finally, single gene deletion cell lines of these candidate genes were constructed, infected with BPIV-3, and the absolute quantification of BPIV-3 genome copy number was performed, and it was found that after deletion of these candidate genes, BPIV-3 replication was significantly inhibited, and after gene complementation, the antiviral effect of the cells disappeared.

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

[0009] 1) The designed sgRNA library contains 23265 protein-coding genes, almost all annotated protein-coding genes in the bovine genome, and covers a wide range. Moreover, four sgRNAs are designed for each gene, targeting the upstream position of the gene open reading frame, which can ensure the gene knockout efficiency, and the maximum allowed mismatch base number of each sgRNA is 5bp, which reduces the off-target risk.

[0010] 2) The cell mutant library can be applied to a variety of viruses, and viruses that can replicate and proliferate in MDBK cells and induce cell death can be screened using it. The sgRNA plasmid library can be subcultured multiple times, and the constructed cell mutant library can also be subcultured, which can ensure that the sgRNA content and coverage will not deviate greatly.

[0011] The second object of the present application is to provide WNT5A gene screened by the above-mentioned method in regulating host anti-BPIV-3 function. The third object of the present application is to provide a reagent for knocking out or silencing WNT5A gene in the preparation of anti-BPIV-3 drugs and the use of the reagent in animal cells or breeds.

[0012] To achieve the above purpose, the present application uses CRISPR / Cas9 gene editing technology to construct a targeting vector targeting the WNT5A gene of MDBK cells, and establishes a WNT5A gene knockout cell line and an anti-BPIV-3 virus infection cell model through vector transfection and cell screening. The results of virology detection show that the WNT5A gene knockout cell shows significant resistance to BPIV-3 infection, and the proliferation rate of the virus on the gene knockout cell is significantly lower than that on the wild type cell, indicating that at the cellular level, WNT5A gene knockout effectively inhibits the infection and proliferation of the virus, and the complementation of WNT5A gene leads to the disappearance of the antiviral effect of the cell, indicating that WNT5A gene plays an important role in participating in BPIV-3 replication and regulating the host's antiviral ability. The present application uses the CRISPR / Cas9-mediated gene knockout method to establish an effective cell model against BPIV-3 infection, confirms the role of WNT5A gene as a target in antiviral infection, and the strategy can be applied to the preparation of WNT5A gene modified anti-BPIV-3 infection cells and animals and the research and development of new drugs.

[0013] The fourth object of the present application is to provide an anti-BPIV-3 drug. The fifth object of the present application is to provide a bovine kidney cell line with WNT5A gene knocked out or silenced.

[0014] To achieve the above-mentioned purpose, the application uses CRISPR / Cas9 gene editing technology to knock out the WNT5A gene of MDBK cells, and finds 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 WNT5A gene can be used to prepare an anti-BPIV-3 drug and an animal cell or breed resistant to BPIV-3 infection. Further, the CRISPR / Cas9 reagent comprises a Cas9 gene editing protein or an expression vector thereof, and an sgRNA or an expression vector thereof for guiding the Cas9 gene editing protein to specifically bind to the WNT5A gene. However, the application is not limited to the reagent developed based on the CRISPR / Cas9 technology, and other reagents for knocking out and silencing the target WNT5A gene designed by using other gene knockout or silencing methods known in the art are also within the protection scope of the application.

[0015] The sixth object of the present application is to provide a preparation method of the bovine kidney cell line with WNT5A gene knocked out or silenced.

[0016] The drug contains a reagent for knocking out or silencing the WNT5A gene. Further, the reagent comprises a Cas9 gene editing protein or an expression vector thereof, and an sgRNA or an expression vector thereof for guiding the Cas9 gene editing protein to specifically bind to the WNT5A gene, and is not limited thereto. The application has verified that the drug can effectively knock out or silence the WNT5A gene of a host cell at the cell level, and further inhibit the infection and proliferation of a virus, and on this basis, it is also feasible to use the drug to edit the genes of other cells or animals, especially a cow, and achieve the same function.

[0017] Figure 1 The bovine kidney cell has the ability of resisting BPIV-3, and also has application prospects in virus isolation and identification and antiviral host factor research.

[0018] Figure 2 Figure 3 The method comprises the following steps:

[0019] (1) designing an sgRNA sequence and primers thereof for the WNT5A gene, and constructing an sgRNA expression plasmid;

[0020] (2) packaging the sgRNA expression plasmid by using a lentiviral vector;

[0021] (3) infecting bovine kidney cells containing a Cas9 protein with the lentiviral vector packaging the sgRNA expression plasmid, to obtain a polyclonal bovine kidney cell line with the WNT5A gene being knocked out;

[0022] (4) selecting a monoclonal cell from the polyclonal bovine kidney cell line.

[0023] The NCBI accession number of the WNT5A gene is GenBank accession no: 530005, and the encoded protein sequence is shown as SEQ ID NO. 1.

[0024] The application successfully constructs a WNT5A gene knockout bovine kidney cell by using a CRISPR / Cas9 gene editing method, and the test results show that the cell can resist BPIV-3 infection, inhibit the titer and expression level of the virus, and the method has high reproducibility.

[0025] The application has the following advantages:

[0026] 1) The application identifies the function of the WNT5A gene at the cell level through functional gain and loss tests, and for the first time finds that the gene can regulate the anti-BPIV-3 infection ability of the host, can be used as a potential target for gene editing animal design, and can be applied to the prevention and treatment of bovine anti-BPIV-3 infection, which has great significance for reducing drug abuse and avoiding the occurrence of an epidemic.

[0027] 2) The WNT5A gene is expected to be used as a target for drug design for candidate compound screening against BEV infection, adding a test compound to a cell model and detecting the expression amount of the CPA6 gene in the cell, and screening out a test compound that can significantly reduce the expression amount of the CPA6 gene.

[0028] 3) The application successfully constructs a WNT5A gene knockout MDBK cell line by using a CRISPR / Cas9 gene editing technology, and the method has the advantages of simplicity, fastness, low cost, high editing efficiency, and can be used for research on BPIV-3 infection related host factors.

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

[0030] Figure 4 : Sequencing statistical analysis of the MDBK cell mutant library. A. MAGeCK analysis of the sequencing results of the cell mutant library; B. The number of sgRNAs of each gene in the cell mutant library.

[0031] Figure 5 : Enrichment gene analysis of the third and fourth screening results. A. KEGG and GO analysis of the top 50 genes in the third and fourth screening; B. KEGG and GO analysis of the top 100 genes in the third and fourth screening. BPIV-3_3rd: the third screening result of the cell mutant library using BPIV-3; BPIV-3_4th: the fourth screening result of the cell mutant library using BPIV-3.

[0032] Figure 6: Genotype of WNT5A gene single deletion clonal cell lines. WT: wild type MDBK; "-" represents the deleted base; red represents sgRNA sequence; green represents PAM sequence.

[0033] Figure 7 : Relative quantitative PCR results after WNT5A gene knockout. ****p<0.0001, mean ± SEM (n=3).

[0034] Figure 8 : EDU cell proliferation level verification results of WNT5A gene knockout cells. WT: wild type MDBK, ns: no significant difference, mean ± SEM (n=3).

[0035] Figure 9 : TCID 50 verification of WNT5A gene deletion cells against virus. WT: wild type MDBK, ns: no significant difference, *p<0.05, **p<0.01, ***p<0.001, mean ± SEM (n=3).

[0036] Figure 1 : Absolute quantitative qPCR verification of WNT5A gene deletion cells against virus. WT: wild type MDBK, ****p<0.0001, mean ± SEM (n=3).

[0037] Figure 2 : Relative quantitative PCR verification results of WNT5A gene complementation. WT: wild type MDBK, ***p<0.001, ****p<0.0001, mean ± SEM (n=3).

[0038] Figure 2 : TCID 50 phenotype verification of WNT5A gene complementation cells to expand BPIV-3. WT: wild type MDBK, ns: no significant difference, ***p<0.001, mean ± SEM (n=3). DETAILED DESCRIPTION

[0039] The technical solutions of the present application are described in further detail below in connection with specific examples. It should be understood that the following examples are only used to illustrate the present application and are not used to limit the protection scope of the present application. Various modifications or equivalent replacements made by those skilled in the art on the basis of the following examples should also be considered to fall within the protection scope of the present application. The experimental methods not specified in the following examples are generally carried out according to the conventional conditions or the reference books such as "Molecular Cloning Experiment Guide" (4th edition), or according to the methods suggested in the operation manual provided by the manufacturer. The materials not specified in the examples, such as MDBK cells, 293T cells, packaging plasmids, T vectors, etc., are all common materials well known in the art, which can be constructed by oneself according to the literature reports or obtained through commercial channels.

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

[0041] 1. Revival and subculture of MDBK cell line containing Cas9 protein

[0042] First, take out the MDBK cells containing Cas9 protein (MDBK-Cas9) previously stored in the laboratory from liquid nitrogen and quickly put them into a 37℃ water bath, constantly shaking during the process to thaw the cells. Place the thawed cells on a sterile operation table, use a pipette to first suck 5mL of cell culture medium (DMEM containing 5% FBS and 2% double antibody), then suck the cell suspension, repeatedly blow for several times, and inject into a centrifuge tube. Centrifuge at 1000r / min for 5min, discard the supernatant. Take 2mL of cell culture medium and transfer the cells to a six-well plate after repeatedly blowing and uniformity. Place in a 37℃, 5% CO2 incubator.

[0043] When the cells are about 95% to 100% full, subculture the cells. The specific operation steps are as follows: take out the six-well plate full of MDBK-Cas9 cells from the incubator, discard the old culture medium, add 1mL of PBS for washing, discard the PBS, add 1mL of trypsin, and incubate at 37℃ in a 5% CO2 incubator for 3min. Then observe whether the cells have detached, add an appropriate amount of cell culture medium and repeatedly blow for more than 10 times, transfer to a 15mL centrifuge tube, centrifuge at 1000r / min for 5min, discard the supernatant. After mixing evenly by blowing 6mL of cell culture medium, transfer to 3 wells of a six-well plate and return to the incubator for continuous culture.

[0044] 2. Lentivirus packaging sgRNA plasmid library

[0045] Recovery and culture: the 293T cells were recovered in a 10 cm cell culture dish, and after full growth, they were passaged. The 3rd generation of 293T cells (10 cm cell culture dish, 90%-95% confluence, fresh DMEM medium containing 10% FBS without antibiotics) in good growth condition were used for lentivirus packaging.

[0046] Transfection: before transfection, the old medium was removed and replaced with DMEM medium containing 5% FBS and 1% penicillin-streptomycin double antibody, and then returned to the incubator (for cells with more floating cells, consider washing with PBS once). For a 10 cm cell culture dish, the total amount of plasmid for lentivirus packaging was 24 μg, of which (PMD2.G: PSPAX2: sgRNA plasmid library = 1:2:3). The corresponding volume of plasmid was added to 500 μL Jetprime Buffer, gently mixed, then 40 μL Jetprime Regent was added, gently mixed, and incubated at room temperature for 10 min. Then it was added to the culture dish and gently shaken before returning to the incubator. After 4-6 h of virus packaging, 10 ml of DMEM medium containing 5% FBS and 1% double antibody was added, and the culture was continued in the incubator. After 24 h, 10 ml of DMEM medium containing 5% FBS and 1% double antibody was added, mixed well, and then returned to the incubator.

[0047] Ultrafiltration: after 60-72 h of virus packaging, the cell morphology was observed, and the cell supernatant was collected (2 10 cm cell culture dishes in one 50 mL centrifuge tube). After sealing with a sealing film, it was centrifuged at 3000 rpm / min for 10 min at 4°C. The supernatant was filtered through a 0.45 μm filter into an ultracentrifuge tube, and then ultracentrifuged at 30000 rpm, 4°C for 2.5 h. Then the supernatant was poured out, and the residual liquid was absorbed on the water-absorbing paper. Each tube was resuspended with 120 μL of pre-cooled PBS, and the same virus concentrate was transferred to the same collection tube after complete blowing and mixing. According to the experimental requirements, the amount was divided and stored at -80°C.

[0048] 3. sgRNA lentivirus library infection of cell line library cells for drug screening to obtain MDBK mutant library

[0049] The concentrated lentivirus was infected with different volumes of MDBK-Cas9 cells (1 x 10 6 Determination of lentivirus titer. Using flow cytometry, 20 μL of lentivirus can achieve a lentivirus infection efficiency of 32.1% in 1 x 10 6

[0050] Next, the MDBK-Cas9 cells were infected with lentivirus at an MOI of 0.3, and a total of 1.4 x 10 8 ​One cell can make the sgRNA coverage of the mutant library reach 500x. On the second day after infection, positive cells were screened by puromycin. After 7 days of screening, the genomes of 1x10 7 Figure 3

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

[0052]

[0053]

[0054] Table 2 PCR amplification reaction system

[0055]

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

[0057] First, we explored the best infection ratio of BPIV-3 to MDBK-Cas9 cells. The virus was diluted by 2-fold, and the cells were infected at an infection ratio of 0.5, 0.05, 0.005, and 0.0005, respectively, with a negative control. The cytopathic effect was observed 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] After that, we used BPIV-3 at an MOI of 0.005 to screen the cell mutant library. After 15 days of screening, many cell colonies could be seen in the culture bottle. These cells were collected and cultured, and a part was stored in liquid nitrogen, and the other part of the cells continued to be screened, and the process was repeated three times, but the MOI gradually increased. After the third round of screening and a part of the surviving cells after the fourth round of screening, the genomes were extracted as templates for PCR, and the high-throughput sequencing primer sequences were as shown in Table 1, and the PCR reaction system was prepared according to Table 2. The PCR products were purified, mixed uniformly, and then sent to the company for high-throughput sequencing. The MAGeCK software was used to analyze the enrichment of sgRNA in the sequencing results, and then the enrichment of genes was inferred. ​​

[0059] 5. Enrichment gene analysis

[0060] KEGG and GO analysis were performed on the top 50 genes screened in the third and fourth rounds, and the results are shown in Figure 4 A. The enriched genes were mainly concentrated in metabolism, positive regulation of IL-6 production, lung development, negative regulation of adipocyte differentiation, etc. Analysis of the top 100 genes screened twice, the results are shown in Figure 5 B. The enriched genes were mainly in the pathways of metabolism, exosome, Golgi apparatus, etc. After 4 rounds of screening with gradually increasing MOI, the screening results have gradually stabilized.

[0061] Example 2: Construction and verification of WNT5A gene knockout cell line

[0062] 1. Construction of WNT5A gene knockout cell line

[0063] Select the top-ranking WNT5A gene from the sequencing results of the fourth round of screening to construct a single gene knockout cell line to further verify whether the gene is involved in the replication of BPIV-3. Connect the sgRNA targeting the gene to the plasmid vector, package the lentivirus with the auxiliary plasmid, and infect the MDBK-Cas9 cells. The sgRNA sequence and plasmid construction primer sequence information are shown in Table 3. Single clones were selected by limiting dilution, expanded and cultured, and the cell genome was extracted as a template for PCR amplification. The PCR amplification primer sequence is shown in Table 4, and the genotype was finally determined by sequencing. The results are shown in Figure 6 It was found that the WNT5A gene knockout MDBK cells lost a 150 bp fragment.

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

[0065]

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

[0067]

[0068] 2. Verification of the transcription level of the WNT5A gene knockout cell line

[0069] RNA was extracted from wild-type MDBK and WNT5A-mutant gene knockout monoclonal cell lines using the Trizol method, and 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 7 As shown, compared with wild-type MDBK cells, the WNT5A-mutant gene knockout monoclonal cell line 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 WNT5A gene-deficient cells

[0076] To assess cell proliferation levels after knockout of candidate genes, wild-type MDBK and knockout cell lines designated WNT5A-mutant 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 with 5% CO2. After 24 hours of incubation, cells were transferred to BeyoClick microplates containing Alexa Fluor 555 (company: Beyotime; 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 8 As shown, the WNT5A gene mutant cells did not show a significant change in proliferation capacity compared to wild-type MDBK cells, and this cell line can be used for subsequent validation experiments.

[0077] Example 3: Using TCID 50Detection of anti-BPIV-3 ability of WNT5A gene-deleted MDBK monoclonal cells by absolute quantitative qPCR

[0078] 1. TCID of WNT5A gene-deficient cells resisting BPIV-3 virus 50 verify

[0079] Wild-type MDBK and WNT5A 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 WNT5A gene-deleted cell line samples was detected. 50 phenotypic differences were observed. The results were as follows: Figure 9 As shown, after infection with BPIV-3, the viral titer of the WNT5A gene-deleted cell line was significantly lower than that of wild-type MDBK.

[0080] 2. Absolute quantitative qPCR verification of WNT5A gene deletion 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: ​ As shown, the same as TCID 50 The results were consistent; compared to wild-type MDBK, the BEV virus copy number expanded in the WNT5A gene-deleted cell line was significantly reduced. This result demonstrates that the WNT5A 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 WNT5A gene complementation

[0085] 1. Construction of WNT5A gene complementation plasmid

[0086] The wild type MDBK cell extracts RNA, and after reverse transcription to obtain cDNA, the WNT5A gene is amplified with it as a template, and the Flag tag is added before the terminal stop codon of the target sequence; the amino acid mutation near the NGG of sgRNA is changed to a synonymous amino acid, and the WNT5A backfill sequence is obtained. The pLVX-EGFP-IRES-Neo vector is double enzyme cut by EcoRI (company: New England Biolabs; number: R0101V) and BamHI (company: New England Biolabs; number: R0136V), and the 8269bp vector band is recovered. The homologous recombination enzyme (company: Nanjing Novozyme Biotech Co., Ltd.; number: C112-01) is used to connect the target band and the vector band, and the pLVX-c WNT5A-flag backfill plasmid is constructed. 4°C connection overnight, pick bacteria identification, sequencing to obtain pLVX-c WNT5A-flag backfill plasmid. According to the plasmid extraction kit (company: Omega Bio-Tek; number: D6950-02), the endotoxin-free plasmid is extracted and stored at -80°C. The enzyme cutting reaction system of pLVX-EGFP-IRES-Neo vector is as shown in Table 8, the enzyme cutting connection system of backfill sequence fragment and vector is as shown in Table 9, and the homologous recombination amplification primer sequence is as shown in Table 10.

[0087] Table 8 Enzyme cutting reaction system

[0088]

[0089] Table 9 Backfill sequence fragment and vector enzyme cutting connection system

[0090]

[0091]

[0092] Table 10 Homologous recombination amplification primer sequence information

[0093]

[0094] 2. Obtaining of WNT5A gene backfill cell line

[0095] The method of step 2 in Example 1 is used for slow virus packaging pLVX-c WNT5A-flag backfill plasmid, that is, the "sgRNA plasmid library" in the step is replaced by the pLVX-c WNT5A-flag backfill plasmid in step 1 above.

[0096] The WNT5A-mutant cell line is recovered in a six-well plate, and after the plate is fully covered, the cells are subcultured at 1:3. When the cells are about 60% full, the old culture medium is discarded, 1 mL of cell maintenance solution (containing 2% FBS + 1% double-antibiotic DMEM) is added to each well, 1 μL of polybreme is added, 40 μL of WNT5A complementation plasmid lentivirus is added, and the mixture is mixed and incubated for 24 h. The old culture medium is then discarded, 2 mL of culture medium containing 5% FBS and 1% double-antibiotic is added, and the cells are incubated for another 24 h. Then, 2 mL of DMEM culture medium containing 5% FBS, 1% double-antibiotic and 400 μg / mL of neomycin is added for drug screening. After all the negative control drug screening wells are dead, the positive control and negative control are subcultured (note: the positive control wells are subcultured at 1:2 in a six-well plate, and the negative control is subcultured in two wells, one of which is used as a negative control drug screening group). In this way, the drug screening is performed for a total of 7 days, and the WNT5A gene complemented MDBK cell line is obtained. The total RNA of the complemented cells is extracted using Trizol, and the relative quantification method is used to verify whether the complementation experiment is successful. The results are shown in ​ FIG. 6, and the WNT5A gene complementation is successful.

[0097] 3. Phenotype identification of WNT5A gene complemented cell strain

[0098] The wild-type MDBK cells, WNT5A gene knockout cells and WNT5A gene complemented cells are respectively infected with BPIV-3 virus by referring to Example 3, and the TCID 50 to verify whether the phenotype is restored. The results are shown in ​ FIG. 7, and the susceptibility to BPIV-3 is restored after the WNT5A gene complementation.

Claims

1. Use of a reagent for knocking out or silencing a WNT5A gene in the preparation of a medicine for resisting bovine parainfluenza virus type 3 (BPIV-3), wherein the amino acid sequence encoded by the WNT5A gene is shown as SEQ ID NO. 1, and the reagent comprises a Cas9 gene editing protein and an sgRNA for guiding the Cas9 gene editing protein to specifically bind to the WNT5A gene, wherein the sequence of the sgRNA is shown as SEQ ID NO.

2.

2. A method of preparing an anti-BPIV-3 bovine kidney cell line, characterized by The method comprises the following steps: (1) designing an sgRNA sequence for the WNT5A gene and primers thereof, and constructing an sgRNA expression plasmid; (2) packaging the sgRNA expression plasmid by using a lentivirus vector; (3) infecting bovine kidney cells containing a Cas9 protein with the lentivirus vector packaged with the sgRNA expression plasmid to obtain a polyclonal bovine kidney cell line with the WNT5A gene knocked out; (4) selecting a single clone from the polyclonal bovine kidney cell line with the WNT5A gene knocked out, wherein the amino acid sequence encoded by the WNT5A gene is shown as SEQ ID NO. 1, and the sequence of the sgRNA is shown as SEQ ID NO.

2.

3. The method for preparing the anti-BPIV-3 bovine kidney cell line as described in claim 2, characterized in that: The sequences of the primers are shown as SEQ ID NO. 3 and 4.

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