Anti-Newcastle disease virus sgRNA targeting ATP11c gene, recombinant vector and application

Abstract

The invention provides an ATP11c gene targeting anti-Newcastle disease virus sgRNA, a recombinant vector and an application, and relates to the technical field of gene engineering, the invention provides the ATP11c gene targeting anti-Newcastle disease virus sgRNA based on a CRISPR system, the ATP11c gene can be effectively targeted and knocked out by using the sgRNA provided by the invention, and the sgRNA has the advantages of short operation time, accurate targeting, high sensitivity, low cost and the like. By knocking out the ATP11c gene, the invention discovers that the ATP11c gene knockout cell strain can reduce the Newcastle disease virus infection titer; in addition, a small molecular targeting drug designed for the ATP11c gene can also be applied to inhibition of NDV infection of host cells and NDV susceptible chicken flocks.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to an anti-Newcastle disease virus sgRNA targeting ATP11c gene, a recombinant vector and application. BACKGROUND

[0002] The poultry breeding industry has been threatened by some diseases that can reduce its yield. Among them, the most common and most serious viral diseases of poultry include Newcastle disease (ND), avian influenza (AI), etc. Newcastle disease is a high fever and highly contagious disease caused by Newcastle disease virus (NDV), with high morbidity and mortality. It is also a zoonosis, and humans infected with it can develop conjunctivitis. For decades, Newcastle disease has been considered the top viral disease that limits the development of the poultry industry.

[0003] In the prior art, the prevention and control of Newcastle disease often adopts a strategy of preventive vaccination combined with drug treatment. However, this technology often has the defects of limited protection effect, inconvenience of use, high requirements for storage and transportation, and poor treatment effect. SUMMARY

[0004] To solve the above problems, the present application provides an anti-Newcastle disease virus sgRNA targeting ATP11c gene, a recombinant vector and application.

[0005] ATP11C, as a P4 subtype phospholipid transport ATPase, dynamically maintains the polarity distribution of plasma membrane lipid components by catalyzing the internalization and translocation of negatively charged amino phospholipids in the double-layer membrane structure. The transmembrane localization of phosphatidylserine mediated by this enzyme has a core regulatory role in red blood cell mechanical deformation ability, immune synapse formation and lipid homeostasis balance.

[0006] The present application found that knocking out ATP11c The gene can reduce the titer of Newcastle disease virus infection. Small molecule targeted drugs designed for the host factor ATP11c gene can block the fusion process of Newcastle disease virus (NDV) and host cell membranes, and inhibit the invasion of virus into host cells. In chicken flock trials, the drug can significantly reduce the morbidity and viral load of NDV susceptible chicken flocks.

[0007] To achieve the above purpose, the present application provides the following technical solutions: In a first aspect, the present application provides an anti-Newcastle disease virus sgRNA targeting ATP11c gene, wherein the sgRNA is sgRNA-1F, sgRNA-1R, or sgRNA-2F, sgRNA-2R, or sgRNA-3F, sgRNA-3R. The nucleotide sequence of the sgRNA-1F is shown as SEQ ID No. 1, the nucleotide sequence of the sgRNA-1R is shown as SEQ ID No. 2, the nucleotide sequence of the sgRNA-2F is shown as SEQ ID No. 3, the nucleotide sequence of the sgRNA-2R is shown as SEQ ID No. 4, the nucleotide sequence of the sgRNA-3F is shown as SEQ ID No. 5, and the nucleotide sequence of the sgRNA-3R is shown as SEQ ID No. 6.

[0008] In a second aspect, the present application provides a recombinant vector for knocking out the ATP11c gene, wherein the recombinant vector comprises the sgRNA and the lentivirus sgRNA expression vector according to the first aspect.

[0009] In addition, the lentivirus sgRNA expression vector is a LentiCRISPR v2 lentivirus vector.

[0010] In a third aspect, the present application provides the use of the anti-Newcastle disease virus sgRNA targeting the ATP11c gene: 1) for preparing a cell line product for reducing the titer of Newcastle disease virus infection; 2) for preparing a drug product for resisting Newcastle disease virus infection.

[0011] In a fourth aspect, the present application provides the use of the recombinant vector for knocking out the ATP11c gene: 1) for preparing a cell line product for reducing the titer of Newcastle disease virus infection; 2) for preparing a drug product for resisting Newcastle disease virus infection.

[0012] Advantages: 1) The present application is based on the CRISPR system, and provides an anti-Newcastle disease virus sgRNA specifically targeting the ATP11c gene. ATP11c The sgRNA provided by the present application can effectively target the ATP11c gene and knock out the gene, has the characteristics of short operation time, precise targeting, low preparation cost, etc. ATP11c By knocking out the ATP11c gene, the present application finds that ATP11c The ATP11c gene knockout cell strain can reduce the titer of Newcastle disease virus infection. ATP11c In addition, by knocking out the ATP11c gene, the present application can cultivate ATP11c The ATP11c gene knockout chicken strain has potential application in the research of natural resistance to NDV infection.

[0013] 2、The application provides an anti-Newcastle disease virus sgRNA targeting the ATP11c gene, NDV infection can induce apoptosis of host cells and cause abnormal exposure of inner side phosphatidylserine (PS) of the plasma membrane outside the cell membrane, and the P4-ATPase family member ATP11c serves as a specific phospholipid flippase, is responsible for translocating PS to the cytoplasmic side of the plasma membrane and maintaining the correct distribution of the cell membrane phospholipid, after the ATP11c gene is knocked out, the ATP11c deletion effectively delays the PS outturn process by inhibiting the membrane asymmetry destruction mediated by caspase, thereby weakening the maturation and release of Newcastle disease virus particles, and thus significantly reducing the infection titer.

[0014] 3、The application provides an anti-Newcastle disease virus sgRNA targeting the ATP11c gene, by knocking out the ATP11c gene to cultivate disease-resistant chicken breeds, the mortality of the chicken population is significantly reduced, the economic loss is reduced, the egg production and growth performance are improved, and additional costs such as isolation and culling caused by an outbreak of an epidemic are avoided.

[0015] 4、The application can effectively reduce the infection titer of Newcastle disease virus (NDV) in the DF-1 cell strain by knocking out the ATP11c gene, and experimental results show that at 18 h and 24 h time points, the NDV TCID50 of the ATP11c gene knockout DF-1 cell strain is only 84% of that of the wild type (WT) DF-1 cell, indicating that the virus titer of NDV in the gene knockout cell strain is significantly reduced, and the ability of the cell to be infected is obviously inhibited, thereby providing a new effective strategy for preventing and controlling Newcastle disease.

[0016] 5、The application can significantly reduce the ability of cells to flip PS from the outer side of the membrane to the inner side by knocking out the ATP11c gene, fluorescence microscopy observation shows that the fluorescence intensity of the ATP11c KO cell is significantly lower than that of the WT cell; the fluorescence intensity of the KO cell is only 20% of that of the WT cell, and this result shows that the ATP11c gene knockout causes the ability of the cell to flip PS from the outer side of the membrane to the inner side to be significantly reduced; NDV infection can induce apoptosis of host cells, causing irreversible changes in the distribution of the cell membrane phospholipid bilayer, and in the apoptosis process, the anionic phospholipid-phosphatidylserine (PS) originally located on the inner side of the plasma membrane will be flipped and exposed on the outer side of the plasma membrane; PS flipping is crucial to the virus infection cycle: various enveloped viruses such as HIV, EBOV, DENV, etc., can use the exposed PS as an “eat me” signal, combine with the PS receptor on the surface of immune cells such as TIM and TAM family, promote virus adsorption and internalization, PS flipping is not only related to the apoptosis signal, but also can directly affect the budding and transmission efficiency of the virus particles and regulate the strength of the host anti-virus immune response. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings required to be used in the embodiments will be briefly introduced as follows.

[0018] Figure 1 For DNA sequencing technology to detect the gene knockout results of ATP11c KO cell strains; Figure 2 For Western Blot experiment to detect the ATP11c gene knockout results of ATP11c KO cell strains; Figure 3 For the relative expression amount results of ATP11c gene of WT and ATP11c KO cell strains; Figure 4 For RT-qPCR technology to detect the relative expression amount changes of NDV NP gene transcription level; Figure 5 For Western Blot to detect the NDV NP protein level change results of infected WT cells and ATP11c KO cells; Figure 6 For the relative expression amount results of NDV NP protein of infected WT cells and ATP11c KO cells; Figure 7 For Western Blot to detect the influence results of overexpressed ATP11c protein on NDV NP protein replication; Figure 8 For the relative expression amount results of NDV NP protein of overexpressed ATP11c protein; Figure 9 For the virus titer TCID 50 determination results; Figure 10 For the influence results of ATP11c gene knockout on flip PS. DETAILED DESCRIPTION

[0019] In order to further illustrate the present application, the targeted ATP11c gene anti-Newcastle disease virus sgRNA, recombinant vector and application provided by the present application will be described in detail in combination with the drawings and embodiments, but they cannot be understood as limiting the protection scope of the present application.

[0020] Example 1 Construction of recombinant plasmid: According to the chicken-derived ATP11c gene sequence (Gene ID: 422254) in NCBI GenBank, 2 pairs of 20 bp sgRNA (Single guide RNA) primers oligo DNA were designed for ATP11c gene by CRISPR sgRNA design tool https: / / www.synthego.com. They were named sgRNA1 and sgRNA2, and the primer sequences are shown in Table 1. Then the reverse complementary sequence of the target sequence was obtained by reverse complementing the primer sequence, followed by adding CACCG at the 5' end of the forward sequence (the sequence immediately after NGG, N represents any nucleotide), if the first base of the forward sequence is G, no additional G is needed; add AAAC at the 5' end of the reverse sequence and add C base at the 3' end, if the first base of the forward sequence is G, i.e. the last base of the reverse sequence is C, no additional C is needed. The modified forward and reverse sequences were sent to Shanghai Sangon Biological Engineering Co., Ltd. for oligo synthesis.

[0021] Table 1 sgRNA primer sequence targeting ATP11c gene

[0022] The upstream and downstream sequences of the synthesized sgRNA primer sequence were treated by PCR gradient annealing (to form double-stranded), and the specific reaction conditions were as follows: 1 μL (100 μM) of forward and reverse sequences, 8 μL of ddH2O, and a total system of 10 μL; run in the PCR instrument according to the following program: 37℃, 30 min; 95℃, 5 min; 95℃, 1 min, for 15 cycles; finally, reduce to 4℃, and the PCR sample can be stored at 4℃ for a short time.

[0023] The LentiCRISPR v2 lentivirus vector (Addgene Plasmid #52961) was linearized, and the specific enzyme digestion system was as follows: 1 μg of LentiCRISPR v2 lentivirus vector, 1 μL of BsmBⅠ enzyme (NEB #R0580), 5 μL of NEBuffer, and ddH2O was added to 50 μL; the reaction condition was 55℃ water bath overnight. After enzyme digestion, all samples were subjected to nucleic acid gel electrophoresis, and then the large fragment with a size of about 11 kb was recovered and purified using a gel recovery kit (Tiangen DP209), and the small fragment with a size of about 2 kb was discarded.

[0024] The purified linear vector was connected with the double-stranded sgRNA primer, and the connection system was as follows: 2 μL of double-stranded sgRNA annealing primer, 3 μL of linear vector LentiCRISPR v2, 1 μL of T4 ligase, 1 μL of 10× ligase buffer, 3 μL of ddH2O, and the total system was 10 μL; the reaction condition was as follows: connection in the connection instrument at 16°C overnight. After the connection was completed, the connection product was transformed into E. coli DH-5α competent cells, and then a single colony was picked and expanded for culture, and then sent to Shanghai Shenguo Company for sequencing. After the bacterial liquid sequencing result was returned, the sequence was compared and found that the sgRNA was connected to the vector, indicating that the recombinant plasmid was successfully constructed, and the plasmids were named as pLATP11c-sgRNA1 and pLATP11c-sgRNA2, respectively.

[0025] Packaging of lentivirus: an appropriate amount of 293T cells were plated in a 10 cm cell culture dish, and cultured for 20 h (at this time, the cell confluence was about 70%) and then transfected. The plasmid system for transfection was 6 μg of recombinant plasmid (pLATP11c-sgRNA1, pLATP11c-sgRNA2, respectively), 8 μg of pxPAX2 helper plasmid (Addgene, Plasmid #12260) and 2 μg of pMD2.G helper plasmid (Addgene Plasmid #12259), the transfection reagent was 50 μL of PolyethylenimineLinear (PEI) MW40000 (NoninBio NBS4000), and the total transfection system was 2 mL, then 8 mL of Opti-MEM medium (Thermo Fisher product, catalog number: 11058021) was added to the culture dish to make up to 10 mL, and the cell supernatant was collected after 60 h of transfection and filtered with a 0.45 μm microporous filter. At this time, the obtained filtrate was lentivirus, named ATP11c-sgRNA1, ATP11c-sgRNA2, and ATP11c-sgRNA3. The lentivirus was divided and stored at -80°C for standby.

[0026] Determination of the optimum concentration of puromycin: an appropriate amount of DF-1 cells were plated in a 12-well plate, and cultured for 12 h, then replaced with DMEM medium containing working concentrations of 0, 1, 1.25, 1.5, 2, 2.5, 3.5, 5 and 10 μg / mL of puromycin and 10% FBS, and then cultured for 48 h. The survival state of the cells was observed, and the lowest drug concentration that could kill all the cells was selected as the optimum concentration of puromycin for screening cells, i.e. 3.5 μg / mL.

[0027] Infection of lentivirus and drug screening experiment: the DF-1 cells were inoculated in a 6-well plate, and after 12 h of culture, they were infected with lentivirus ATP11c-sgRNA1 and ATP11c-sgRNA2, respectively. One well was added with 1 mL of lentivirus and 1 mL of blank DMEM medium mixture, and the other well (negative control well) was added with 2 mL of blank DMEM medium. Each well was added with 2 μL of polybrene (10 mg / mL) to promote the adsorption of lentivirus. After 12 h of infection, the supernatant was discarded, and the cells were cultured in DMEM medium containing 2% FBS for 36 h. Then the cell supernatant was discarded, and DMEM medium containing 3.5 μg / mL of puromycin and 10% FBS was added for drug screening experiment. The medium containing puromycin was replaced every two days, and the cells were cultured for six days until the cells in the negative control well died. At this time, only a few cells survived in the well infected with lentivirus. The cells were washed with PBS for 3 times, and the medium was replaced with DMEM medium containing 2% FBS (without puromycin). The cells were cultured in the incubator, and the state of the surviving cells was observed every day.

[0028] Obtaining a monoclonal cell line: the cells were blown apart after trypsin digestion, and 20 μL of the cells were taken for counting with a cell counting plate. Then a small part of the cells was diluted to about 1 cell per well of a 96-well plate, i.e. less than 100 cells in 10 mL of diluent. Then the diluted liquid was added to the 96-well plate at 100 μL / well. After the cells grew from one to a group of cells, the cells were digested and transferred to a 48-well plate, and then to a 12-well plate until the cell amount could be inoculated. At the same time, a control group (WT) was set, i.e. DF-1 cells not infected with lentivirus.

[0029] Detection of knockout efficiency: 1. The monoclonal cells were digested with trypsin and the digestion was terminated with 1 mL of 10% FBS DMEM medium. The cell suspension was transferred to a 2 mL EP tube, and DNA was extracted from the monoclonal cells using a cell genomic DNA extraction kit (Tiangen DP304). The DNA samples could be briefly stored at -20℃. Primers were designed approximately 200 bp upstream and downstream of two pairs of sgRNA sequences, named ATP11c-KO-F and ATP11c-KO-R. The forward and reverse sequences were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are shown in Table 2. PCR amplification was performed using the extracted DNA as a template. The high-fidelity enzyme used was PhantaMax Super-Fidelity DNA Polymerase (Novizan P505). The PCR system consisted of: 2 μL DNA, 17 μL ddH2O, 25 μL 2×PhantaMax Buffer, 1 μL dNTP, 2 μL each of primers ATP11c-KO-F and ATP11c-KO-R (sequences shown in Table 2), and 1 μL PhantaMax Super-Fidelity DNA Ploymerase, for a total volume of 50 μL. The PCR instrument was run according to the following program: 95℃ for 3 min; 95℃ for 15 s; 60℃ for 15 s; 72℃ for 1 min; 72℃ for 5 min; and finally, cooled to 4℃; Steps 2-4 were repeated for 34 cycles. All PCR samples were subjected to nucleic acid gel electrophoresis, and the target fragment of approximately 500 bp was recovered and purified using a gel extraction kit (Tiangen DP209). The gel-recovered product was ligated into a T-vector for sequencing. First, the gel-recovered product was mixed with Taq enzyme at a 1:1 ratio and incubated at 72°C for 30 min. The mixture was then liquid-gel recovered and ligated using the following mixture: 2.5 μL Solution I, 0.25 μL 19T, and 2.25 μL gel-recovered product, for a total volume of 5 μL. The mixture was incubated in a 16°C metal bath for 1-2 h. After ligation, the ligation product was transformed into *E. coli* DH-5α competent cells. Single colonies were then picked, expanded, and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequence alignment revealed deletions of 23 bp and 13 bp near the ATP11c-sgRNA1 and ATP11c-sgRNA2 sequences, respectively. The presence of only these two deletions indicates successful ATP11c gene knockout. The results are as follows: Figure 1 As shown.

[0030] Table 2 Primer sequences for ATP11c-KO-F and ATP11c-KO-R

[0031] 2. After scraping the above monoclonal cells and transferring them to EP tubes, centrifuge (1000 rpm / min), discard the supernatant, wash the cells 3 times with PBS, and then add 200 μL of 2×SDS-PAGE protein loading buffer to each well of a 6-well plate. Then, use a 1 mL pipette tip to rotate the sample in one direction to form a ball and aspirate it into a 1.5 mL EP tube. Boil the sample in a metal bath at 100℃ for 10 min. After boiling, briefly centrifuge the liquid on the EP tube and store the protein sample at -20℃ for later use.

[0032] SDS-PAGE gel electrophoresis: Add the sample to different lanes (note whether there are any gel blocks blocking the lanes). After adding the sample, close the electrophoresis tank lid (note that the positive and negative electrodes should not be reversed). First, use a constant voltage of 80 V for about 30 minutes. When the sample is pressed into a straight line and enters the separating gel from the stacking gel, the voltage can be adjusted to 120 V and continue constant voltage electrophoresis for about 70 minutes. When the bromophenol blue band of the sample moves down to the bottom of the separating gel, the electrophoresis can be stopped. At this time, the electrophoresis is complete.

[0033] Western Blot Experiment: Before the transfer process, prepare a 5% skim milk solution with 1×TBST and shake it on a shaker. After transfer, discard the gel and place the NC membrane in the 5% skim milk solution for blocking and incubation at room temperature for 2 hours. After blocking, discard the skim milk solution, add 1×TBST, and gently shake on a shaker to wash the membrane. After 5 minutes, discard the original liquid and add fresh 1×TBST to continue washing, repeating this process three times. After washing, incubate with rabbit anti-ATP11c antibody (antibodies A88371) at 4°C overnight. After primary antibody incubation, wash the membrane with 1×TBST on a shaker for 10 minutes each time, repeating three times. Then incubate with goat anti-rabbit secondary antibody (Abcam ab6721) at room temperature for 2 hours, repeating the above washing steps for 10 minutes each time, for a total of three replicates. After washing, an ECL luminescence developing solution was prepared in the dark. The NC membrane was incubated in the developing solution for approximately 30 seconds. The membrane was then scanned and developed using a fully automated chemiluminescence imaging analysis system. The results were saved and analyzed. (See attached table for details.) Figure 2 .

[0034] The results of Western blot analysis were quantitatively analyzed using ImageJ software (a free software developed by the National Institutes of Health). The method is as follows: 1. Open the file; 2. Convert the image to grayscale: Image > Type, select 16-bit; 3. Eliminate background interference: Process > Subtract Background. Selecting 50 should be sufficient. 4. Set quantitative parameters: Analyze > Set Measurements, click on area, average density, grayscale value, and integrated density; 5. Set the unit: Analyze > Set Scale, and enter "pixels" in the box next to "Unit of length"; 6. Convert the image to highlight bars: Edit > Invert; 7. Select Freehand Selection; try to circle the strip; press the 'm' key to display the IntDen grayscale values; 8. Use "Freehand Selection" to select the next strip and press m to take the measurement; 9. After measuring all bands, select "Edit" and then "Select All" in the results. Then copy the data "IntDen" to an Excel spreadsheet for analysis. See the results below. Figure 3 .

[0035] Depend on Figure 2 and Figure 3 It can be seen that, compared with wild-type (WT) cells, the ATP11c protein in DF-1 cells after gene knockout of ATP11c cannot be expressed normally, proving that a new strain has been successfully obtained. ATP11c DF-1 gene knockout cells were named ATP11c KO DF-1.

[0036] Example 2 ATP11c Gene knockout cell lines ( ATP11c Effect of KO DF-1 on Newcastle disease virus proliferation: WT DF-1 cells and ATP11c gene knockout DF-1 cells obtained in Example 1 were compared. ATP11c KO DF-1 cells were plated in 6-well plates, and NDV (Mukteswar strain derived from Yangzhou Weike vaccine strain of Sinopharm Animal Health, [Approval Number] Veterinary Drug Production Permit (2010) 101042003) infection group and virus-free control group were set up. After 1 MOI NDV infection for 1 h, the DMEM medium was replaced with 2 mL of DMEM medium containing 2% FBS. Cell samples were collected at 12 h and 24 h after infection.

[0037] Cellular RNA was extracted, and the relative expression level of the NDV NP gene transcription was detected using real-time quantitative PCR (RT-qPCR). The mRNA transcription level of β-actin, an internal reference gene from chicken DF-1 cells, was selected as the reference for the target gene to reduce errors caused by inconsistent cell numbers. The relative quantitative qPCR reaction system is shown in Table 3, and the RT-qPCR primers are shown in Table 4. A mixture of all components except cDNA was prepared according to the experimental system. After vortexing and mixing, 19 μL was added to each well of the quantitative PCR tube, and finally, the cDNA template was added separately. Each sample was performed in triplicate. The specific RT-qPCR reaction conditions were: 95℃ pre-denaturation for 5 min; then 95℃ denaturation for 10 s, 60℃ annealing for 20 s, and 72℃ extension for 20 s, for 40 cycles. The results are shown in Table 3. Figure 4 .

[0038] Table 3 RT-qPCR system

[0039] Table 4 RT-qPCR Primers

[0040] Depend on Figure 4 It can be seen that at the 12-hour time point, ATP11c The NP gene copy number in KO DF-1 cells was approximately 10% of that in WT DF-1 cells at the 24-hour time point. ATP11c The NP gene copy number in KO DF-1 cells was approximately 50% of that in WT DF-1 cells, with a significant difference in transcriptional levels (p<0.05). This indicates that, relative to WT DF-1 cells, NDV infection... ATP11c When KO DF-1 cells are used, the transcription of the NP gene is suppressed.

[0041] Changes in NDV NP protein levels were detected by Western blotting. Results are shown below. Figure 5 The results of Western blot analysis were quantitatively analyzed using ImageJ software. (See attached image.) Figure 6 The results showed that at the 12-hour time point, ATP11c The expression level of NDV NP protein in the gene knockout DF-1 cell line was 76% of that in WT DF-1 cells, with no significant difference at 24 h. NDV in ATP11c The replication capacity of gene knockout cell lines was significantly lower than that of WT cells, indicating that NDV replication within cells was significantly inhibited (p<0.05).

[0042] Example 3 Construction of ATP11c recombinant plasmid: According to the GenBank published chicken ATP11c (NCBI Reference Sequence: XM_420240.8) download the corresponding nucleotide sequence, ATP11c gene was amplified by PCR with the primers in Table 5, and the reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 90 s, repeated for 34 cycles; 72℃ extension for 5 min; 4℃ to end. The reaction system is shown in Table 6. The PCR amplification product was recovered by 1% agarose nucleic acid gel electrophoresis, and the gel recovery kit was used for gel recovery.

[0043] Table 5 ATP11c primer sequence

[0044] Table 6 ATP11c gene PCR amplification system

[0045] The pCMV-HA vector was double digested with EcoR I and Kpn I, and the enzyme digestion system and reaction conditions were as follows: EcoR I 2 μL, Kpn I 2 μL, 5×Buffer 5 μL, pCMV-HA vector 2 μg, ddH2O to 50 μL; 37℃ enzyme digestion for 1 h. The homologous recombination system and reaction conditions were as follows: 2×ClonExpress Mix 5 μL, PCR gel recovery product 2 μL, linearized pCMV-HA vector 1 μL, ddH2O to 10 μL; the homologous recombination reaction conditions were as follows: 55℃ metal bath for 15 min.

[0046] The homologous recombination ligation product was transformed into E. coli DH-5α competent cells, 3-5 single colonies were picked and added to 1 mL of liquid LB medium with ampicillin resistance, and placed in a 37℃ constant temperature incubator for 6 h of expansion culture, then 200 μL of bacterial liquid was taken to a sequencing company for sequencing, and the correctly sequenced recombinant plasmid pCMV-HA-ATP11c was selected for expansion culture and plasmid extraction.

[0047] Using a similar method to Example 2, after transfection of the ATP11c recombinant plasmid pCMV-HA-ATP11c and the control empty vector plasmid pCMV-HA, the cells were infected for 1 h, then the cell culture medium was replaced with 2 mL of DMEM medium containing 2% FBS, and the cell samples were collected at 12 h and 24 h after infection.

[0048] The Western Blot experiment detected the expression level change of NDV NP under the condition of overexpression of ATP11c protein and empty vector plasmid, and the results are shown in Figure 7The results of Western blot analysis were quantitatively analyzed using ImageJ software. (See attached image.) Figure 8 The results showed that at 12h time point, compared with the empty vector control group, the ATP11c protein overexpression group significantly increased the expression level of NP protein by 1.8 times, which was statistically significant (p<0.05). At 24h time point, there was no significant difference between the ATP11c protein overexpression group and the empty vector control group.

[0049] Example 4 Using TCID 50 Methods for determining NDV titers: NDV was used to infect DF-1 cells and ATP11c DF-1 gene knockout cells ( ATP11c KO DF-1 cells were collected at 6, 12, 18, and 24 hours later to determine the viral titer. The specific operating steps are as follows: BHK cells were prepared at a concentration of 1×10⁻⁶ cells / mL. 4 Cells were seeded per well into 96-well cell culture plates and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2 for approximately 12 hours.

[0050] Take 11 EP tubes and add 900 μL of serum-free DMEM medium sequentially. Using a sterile pipette tip, add 100 μL of cell supernatant to the first EP tube, vortex to mix, and then transfer 100 μL of the diluent to the second EP tube. Mix again, and then transfer 100 μL of the diluent to the third EP tube, and so on, performing consecutive 10-fold dilutions, i.e., from 10... -1 Dilute to 10 -11 .

[0051] When the confluence of BHK cells in the 96-well plate reached approximately 80%, the culture medium was discarded, and the cells were washed three times with PBS. The pre-diluted virus buffer was then seeded into the cell culture plate at a volume of 100 μL / well, with each dilution seeded in one longitudinal row, resulting in eight replicates. The last longitudinal row served as a negative control. The 96-well plate was placed in a cell culture incubator for further culture. One hour after seeding, the medium was changed by removing the culture medium and adding 2% FBS in DMEM medium. After 72 hours, BHK cytopathic effects were observed and the results recorded. The TCID of NDV was calculated using the Reed-Muench method. 50 .

[0052] See results Figure 9 The results showed that at 18 h and 24 h time points, ATP11c DF-1 gene knockout cell line NDV TCID 50 It accounted for 84% of WT DF-1 cells, indicating that NDV in ATP11cThe virus titer in the gene knockout DF-1 cell strain is lower than that in the WT DF-1 cell, and the ability of NDV to infect cells is inhibited. It can be seen that, ATP11c The gene knockout can reduce the NDV infection titer.

[0053] Although the above embodiment describes the present application in detail, it is only a part of the embodiment of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

[0054] Example 5 Effect of ATP11c gene knockout on PS flip: Wild type (WT) and ATP11c knockout (KO) DF-1 cells were inoculated in 6-well plates for 24 hours. After the cells adhered, 1 mL of serum-free DMEM medium containing 1 μM NBD-PS was added to each well, and incubated for 1 hour in the dark. After incubation, 200 μL PBS was used to gently wash 3 times to completely remove the uninternalized NBD (nitrobenzoxadiazole) fluorescently labeled PS (NBD-PS). Finally, the cells were covered with an appropriate amount of PBS and immediately observed under a fluorescence microscope.

[0055] Fluorescence microscope observation showed that the fluorescence intensity of ATP11c KO cells was significantly lower than that of WT cells Figure 10 ). Statistical analysis showed that the fluorescence intensity of KO cells was only 20% of that of WT cells (p<0.01). This result shows that ATP11c gene knockout significantly reduces the ability of cells to flip PS from the outer side of the membrane to the inner side.

Claims

1. An anti-Newcastle disease virus sgRNA targeting the ATP11c gene, characterized in that, The sgRNA is sgRNA-1F, sgRNA-1R, or sgRNA-2F, sgRNA-2R, or sgRNA-3F, sgRNA-3R. The nucleotide sequence of the sgRNA-1F is shown as SEQ ID No. 1, and the nucleotide sequence of the sgRNA-1R is shown as SEQ ID No.

2. The nucleotide sequence of the sgRNA-2F is shown as SEQ ID No. 3, and the nucleotide sequence of the sgRNA-2R is shown as SEQ ID No.

4. The nucleotide sequence of the sgRNA-3F is shown as SEQ ID No. 5, and the nucleotide sequence of the sgRNA-3R is shown as SEQ ID No.

6.

2. A recombinant vector for knocking out an ATP11c gene, characterized by, The recombinant vector comprises the sgRNA of claim 1 and a lentivirus sgRNA expression vector.

3. The recombinant vector for knocking out the ATP11c gene according to claim 2, wherein, The lentivirus sgRNA expression vector is a LentiCRISPR v2 lentivirus vector.

4. The use of the anti-Newcastle disease virus sgRNA targeting the ATP11c gene according to claim 1, characterized in that, A product for preparing a product for reducing the titer of Newcastle disease virus infection.

5. The use of the anti-Newcastle disease virus sgRNA targeting the ATP11c gene according to claim 1, characterized in that, A product for preparing a product for resisting Newcastle disease virus infection.

6. The application of the recombinant vector with the ATP11c gene knocked out as described in claim 2, characterized in that, A product for preparing a product for reducing the titer of Newcastle disease virus infection.

7. The application of the recombinant vector with the ATP11c gene knocked out as described in claim 2, characterized in that, A product for preparing a product for resisting Newcastle disease virus infection.

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