CN114958763B - Method for constructing Vero cell strain with DBN1 gene knocked out and application thereof - Google Patents
Method for constructing Vero cell strain with DBN1 gene knocked out and application thereof Download PDFInfo
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Abstract
The invention provides a method for constructing a Vero cell strain with a DBN1 gene knocked out and application thereof, belonging to the field of molecular biology. The construction method comprises the following steps: finding a Vero cell DBN1 gene target spot, and designing a target sgRNA which comprises sgRNA-DBN1-F and sgRNA-DBN1-R; the PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector is cut by Bbs I endonuclease, and then is connected with a double chain formed after sgRNA-DBN1-F and sgRNA-DBN1-R sequences are annealed to obtain a knockout plasmid; and co-transfecting the knockout plasmid, auxiliary packaging plasmids PSPXA2 and PMD.2G into Vero cells, and screening to obtain a Vero cell strain with the DBN1 gene knocked out. The construction method is efficient, the obtained cell strain improves the proliferation titer of PEDV, and the cost is obviously reduced.
Description
Technical Field
The invention belongs to the field of molecular biology, and relates to a method for constructing a Vero cell strain with a DBN1 gene knocked out and application thereof.
Background
The CRISPR/Cas9 is immune defense evolved by bacteria and archaea against invaded viruses, and single or multiple genes can be knocked out simultaneously by CRISPR-Cas9 gene knockout, and long-term stable passage can be realized. Compared with other gene knockout technologies, the CRISPR/Cas9 technology has the advantages of simple operation, high targeting accuracy, low cost and the like.
Developmentally regulated brain protein (DBN 1) is an actin-binding protein found in the visual cap of chickens and is ubiquitous in axons and dendritic spines of a variety of organisms.
Porcine Epidemic Diarrhea Virus (PEDV) belongs to the genus alphacoronavirus among coronaviruses. PEDV is one of the important pathogens causing pig diarrhea, and pigs at each day age are susceptible and are most harmful to suckling piglets. The current prevention and cure means mainly comprises vaccine immunization, and the clinical application mainly comprises inactivated vaccine and attenuated vaccine. Inactivated vaccines are one of the main vaccines and require a large amount of virus. However, the porcine epidemic diarrhea virus propagation titer in the prior art is not high enough and the cost is high.
Disclosure of Invention
The invention aims to provide a construction method of a Vero cell strain with a DBN1 gene knocked out, the construction method is efficient, the obtained cell strain improves the multiplication titer of PEDV, and the cost is obviously reduced.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a construction method of a Vero cell strain with a DBN1 gene knocked out comprises the following steps:
(1) Finding a target spot of a Vero cell DBN1 gene, and designing corresponding target sgRNA which comprises sgRNA-DBN1-F and sgRNA-DBN1-R;
(2) The PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector is cut by Bbs I endonuclease, and then is connected with a double-stranded molecule formed after sgRNA-DBN1-F and sgRNA-DBN1-R sequences are annealed to obtain a knockout plasmid PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0;
(3) And co-transfecting the knockout plasmid PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0 and lentivirus auxiliary packaging plasmids PSPXA2 and PMD.2G into Vero cells, and screening to obtain a Vero cell strain with the DBN1 gene knocked out.
In the invention, the sequence of the target is shown as SEQ ID NO:1 is shown in the specification; the sgRNA-DBN1-F and sgRNA-DBN1-R sequences are shown in SEQ ID NO: 2-3.
In the present invention, puromycin-containing screening is performed in step (3).
The invention also provides application of the constructed Vero cell strain with the DBN1 gene knocked out in the aspect of culturing PEDV.
In the invention, the Vero cell strain with the DBN1 gene knocked out is cultured, then PEDV is inoculated, and the Vero cell strain is continuously cultured by adopting a maintenance solution.
In the present invention, the maintenance solution is a high-glucose DMEM complete medium containing 7.5ug/mL pancreatin.
Has the advantages that: according to the DBN1 gene knockout method based on CRISPR/Cas9 technology, a section of specific sgRNA is designed to recognize DBN1 gene, and finally expression of the DBN1 gene is abnormal. The Vero cell DBN1 knockout method and the DBN1 gene expression level detection method have strong practicability. The method can be used for specific knockout of DBN1 gene, and has the advantages of high efficiency, simplicity, economy and the like; the research on the DBN1 gene provides important basis for researching and developing novel PEDV resistant medicaments and searching for an effective pig diarrhea prevention and control method; according to the invention, through a Vero-DBN1-KO cell PEDV virus inoculation experiment, mRNA expression, protein expression and virus titer of viruses are determined, and the effect of DBN1 on inhibiting PEDV replication is found for the first time. The DBN1 knockout cell line established improves the proliferation titer of PEDV, obviously reduces the cost, provides a basis for exploring a cell line with high replication level of PEDV, and has an important role in further researching the proliferation of PEDV.
Drawings
FIG. 1 is a map of PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector; the marked is the BbsI enzyme cutting site position, and is located at two positions of 245bp and 267 bp.
FIG. 2 shows the expression of DBN1 protein after PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0 transfects Vero cells in example 1 of the present invention; wherein ctrl: wild type Vero cells (negative control), DBN1-KD: vero cells with DBN1 knockdown.
FIG. 3 shows the expression of DBN1 protein of the selected monoclonal cell line in example 1 of the present invention. Wherein ctrl: wild type Vero cells (negative control), DBN1-KO: vero cells with DBN1 gene knockout.
FIG. 4 shows the results and analysis of gene sequencing of cell lines in which DBN1 protein is not expressed in example 1 of the present invention. Wherein, the rectangle frame is the target position of the sgRNA sequence, and the arrow is the insertion base position.
FIG. 5 shows the level of virus replication when PEDV was propagated using wild-type Vero cells and cell line DBN1-KO-Vero, respectively. Wherein, the abscissa is the number of hours after virus inoculation (hpi), the ordinate is the mRNA expression level of PEDV, P < 0.01, P <0.001, wt: wild type Vero cells (negative control), DBN1-KO: the BN1 gene knockout cell strain DBN1-KO-Vero.
FIG. 6 shows that PEDV is proliferated by wild-type Vero cells and DBN1-KO-Vero cells respectively, and Western bot detects the expression of the DBN1 and PEDV-N proteins, wherein GADPH is an internal reference, hpi, WT and DBN1-KO are the same as those in FIG. 5.
FIG. 7 shows the IFA detection of the expression of N protein when PEDV was propagated using wild-type Vero cells and cell line DBN1-KO-Vero, respectively. DAPI: a nuclear dye. Mock: non-inoculated wild type Vero cells (blank). hpi, WT, DBN1-KO are the same as in FIG. 5.
FIG. 8 shows the results of virus titer measurement by harvesting cell supernatants of PEDV propagated by wild-type Vero cells and DBN1-KO-Vero cells, respectively. Wherein the abscissa is the time after virus inoculation (hpi) and the ordinate is the PEDV viral titer level log (TCID) 50 /ml)。
Detailed Description
The following examples facilitate a better understanding of the invention, but do not limit it. The experimental procedures in the following examples are all conventional biochemical procedures unless otherwise specified. The test materials used in the following examples are all conventional biochemical reagents unless otherwise specified. The quantitative results in the following examples were obtained by setting up three replicates.
Experimental materials: bbs I endonuclease, 6 Xloading buffer solution and SuperSignal TM West Pico ELC chemiluminescence kit, 10 × Standard Taq Reaction Buffer were purchased from Thermo company; the endotoxin-free medium extraction kit and the gel recovery kit are purchased from TIANGEN company; horse radish peroxidase-labeled goat anti-rabbit IgG antibody, horse radish peroxidase-labeled goat anti-mouse IgG antibody and mouse monoclonal antibody GAPDH are purchased from Proteitech Group company; RIPA protein lysate, PMSF protease inhibitor, puromycin, DAPI stain, FITC goat anti-mouse fluorescent antibody purchased from Biyunnan corporation; chamQ Universal SYBR qPCR Master Mix, hiScript II Q RT SupThe erMix for qPCR reverse transcriptase, fastpure cell/Tissue toll RNA Isolation Kit V2 are all available from Vazyme;
PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector plasmid, lentivirus helper packaging plasmids PSPXA2, PMD.2G were purchased from Beijing central office; PEDV-N, rabbit anti-DBN 1 monoclonal antibodies were purchased from Abcam, DNA extraction kits were purchased from OMEGA, T4 DNA Ligase from Invitrogen, newborn bovine serum was purchased from Gbico, and high-sugar DMEM complete medium was purchased from Sorbet Biotech.
Example 1 preparation of Vero-DBN1-KO cells
1. sgRNA design
The exon sequence of DBN1 gene (accession XM _ 008015437.2) of African green monkey (Chlorocebus sabaeus) kidney cells (Vero cells) was found out by NCBI. Screening a Vero cell DBN1 gene target through an sgRNA design website (http:// cruispr. Mit. Edu) to obtain a target (SEQ ID NO. 1) located from 1560 th to 1579 th of a CDS region of a African green monkey DBN1 gene. The sequences of the targets are as follows: 5 'and 3' of TGCCTGAGGGGCTCCTCCA-.
Designing a sgRNA sequence targeting the target, and adding CACCG at the 5' end to obtain a sgRNA-DBN1-F sequence (SEQ ID NO. 2); adding base AAAC at 5 'end and C at 3' end to obtain sgRNA-DBN1-R sequence (SEQ ID NO. 3).
The sequence of sgRNA-DBN1-F is 5,
the sequence of sgRNA-DBN1-R is 5-.
2. Construction of recombinant plasmids
1. Linearized vector
The PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector map is shown in FIG. 1. The PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector was subjected to single-enzyme digestion with Bbs I endonuclease. Enzyme digestion system: restriction enzymes Bbs I1. Mu.L, PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 plasmid 1. Mu.g, 10 XNE Buffer 2uL, ddH 2 O make up the volume to 50. Mu.L. The reaction procedure of enzyme digestion is as follows: the digestion was carried out for 1h at 37 ℃ and stopped by adding 6 XLoading. And (3) carrying out electrophoresis on the enzyme digestion product by using 1% agarose gel, cutting a target band, and carrying out gel recovery on the target band to recover DNA by using a kit.
Annealing of sgRNA fragments
Annealing the sgRNA-DBN1-F fragment and the sgRNA-DBN1-R fragment to form a sgRNA double-stranded molecule with sticky ends at two ends. An annealing system: 10 × Standard Taq Reaction Buffer 2 μ L, sgRNA-DBN1-F (100 uM) 9 μ L, sgRNA-DBN1-R (100 uM) 9 μ L, overall system: 20uL. And (3) annealing conditions: water bath at 95 deg.c for 5min and connection for further use.
3. Connection of
And connecting the sgRNA double-stranded molecules formed after annealing with a Bbs I single digestion product of a PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector plasmid. A connection system: 7 mu L of annealing product, 1.5 mu L of PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 enzyme digestion product, 1 mu L of T4 DNA Ligase Buffer and 0.5 mu L of T4 DNA Ligase, wherein the total amount is 10 mu L; the ligation system was placed in an electronic incubator overnight at 16 ℃.
4. Transformation of
Taking out the Trans5 alpha chemically competent cells from-80 ℃, immediately inserting the cells into ice for thawing, adding the ligation products into the cells when the cells are just thawed, and incubating the cells on ice for 30min; taking out, placing in 42 deg.C water bath, heat-shocking for 45s, placing back on ice, standing for 2min, adding 500uL LB culture medium, and culturing at 37 deg.C and 200rpm for 1h. 200uL, 50uL, and 10uL of the culture medium were aspirated, added to LB containing ampicillin (100. Mu.g/mL ampicillin) dishes, spread evenly on a stick, and incubated at 37 ℃ in an incubator for 12 hours.
5. Screening of the colonies of interest
Selecting single colony for amplification culture, identifying by PCR and gel electrophoresis, further performing amplification culture on correctly identified strains, and extracting plasmids for sequencing by Scirpus organism company. The recombinant plasmid with correct sequencing alignment was designated PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0.
3. Packaging and transfection of lentivirus of targeted Vero cell DBN1 gene
Vero cells were cultured in high-glucose DMEM complete medium containing 8% neonatal bovine serum and 1% diabody (penicillin, streptomycin). Vero cells were cultured at 1X 10 6 One/well was inoculated in 6-well plates. When the cell confluence reaches 70%, recombinant plasmid PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0 and lentivirus helper packaging plasmid PS are mixed by using a Lipofectamine3000 kitPXA2 and pmd.2g were co-transfected into Vero cells. After 36h of transfection, the whole culture medium in 6-well plates was collected and filtered into 1.5mL centrifuge tubes using 0.45 μm filters to obtain packaged lentiviruses, which were stored at-80 ℃.
The Vero cells are paved into 12-well plates, and when the cell confluency reaches 70%, the packaged lentivirus is mixed with high-glucose DMEM complete medium in a volume ratio of 1. Lentivirus infection for 36h, discard medium, PBS washing 3 times. Pancreatin digestion, taking half of cells, continuously plating and passaging, centrifugally collecting the rest cells, and detecting the DBN1 protein expression level by using a Western bot technology.
The Western bot comprises the following specific operation steps:
80uL of RIPA lysate (1% of PMSF included) per well was lysed on ice for 30min, taken up in 1.5mL EP tubes and centrifuged at 12000rpm for 5min. The supernatant was collected and put into a new 1.5ml EP tube, 5 Xloading Buffer was added, treated in a 100 ℃ metal bath for 10min, and the sample was collected and stored at-20 ℃. Samples were run through polyacrylamide gel electrophoresis and the gel-to-PVDF membrane containing the bands of interest was cut. Sealing with 5% skimmed milk powder solution at 60rpm in shaking table for 2h, and washing with PBST for 3 times (each time for 10 min); the mouse monoclonal internal reference antibody GAPDH diluted by 1; respectively incubating with horseradish peroxidase-labeled goat anti-rabbit IgG antibody or horseradish peroxidase-labeled goat anti-mouse IgG antibody diluted by 1; adding SuperSignal TM West Pico ELC chemiluminescence developing solution, gel imaging system imaging, the results are shown in figure 2. As can be seen from FIG. 2, the expression level of DBN1 protein after transfection of the recombinant lentivirus is obviously inhibited, which shows that after the recombinant DBN1 knockout plasmid PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0 carried by the transfected lentivirus, DBN1 gene in partial Vero cells (DBN 1 knock-down Vero cells) is changed, which affects the expression of DBN1 protein, resulting in a lower detection result.
4. Pressure screening and monoclonal identification
Replated DBN1 knockdown Vero cells were cultured normally for 12h, and then medium was changed to medium containing 8ug/mL puromycin for pressure screening. The culture was changed once on the third day, and the culture was continued until day 6 of pressure screening, cells were trypsinized, diluted to 10 cells/mL using the limiting dilution method, and then plated in 96-well plates at 100uL per well. And observing and marking the hole of the single cell, and timely replenishing the liquid. When the cell grows to have a confluence of 50-70%, trypsinizing, carrying out primary-hole passage, and then gradually carrying out expanded culture on 96, 24 and 12-hole plates. And (3) after the cells are transferred into a 12-pore plate, taking part of the cells, and detecting the expression condition of the DBN1 protein by using a Western bot, wherein the Western bot is specifically operated in the third step. As a result, as shown in FIG. 3, it was revealed that the DBN1 protein could not be expressed in the cell line, and it was confirmed that the cell line was a monoclonal cell line in which the DBN1 gene was successfully knocked out.
Selecting cell strains without DBN1 protein expression, extracting total DNA by using a DNA extraction kit, performing PCR amplification by using identification primers DBN1-F and DBN1-R, and cutting a target band after electrophoresis to send to the organism company of the department of Onychidae for sequencing, wherein the result is shown in figure 4. Sequencing shows that the cell strain which does not express the DBN1 protein inserts a base A between the 4 th base and the 5 th base of a target sequence, so that the expression of the DBN1 is abnormal. The successfully identified DBN1 gene knockout cell strain is named as DBN1-KO-Vero. Wherein, the PCR system: primer DBN 1-F1 uL, primer DBN 1-R1 uL, DNA 1uL, 2 XPhanta Master Mix10uL, ddH 2 O make up the volume to 20uL. PCR procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95 deg.C for 30s, annealing at 54.5 deg.C for 30s, and extension at 72 deg.C for 1min for 30 cycles; final extension at 72 ℃ for 10min. The PCR identification primer sequences are as follows:
DBN1-F:5’-AGGGCTGAGGGCAGAGTT-3’，
DBN1-R:5’-GCAGCAGGAAAGAGTCGC-3’。
example 2 examination of the level of PEDV replication in DBN1 knock-out cell line DBN1-KO-Vero
After obtaining a DBN1 knock-out cell strain DBN1-KO-Vero, in order to investigate the influence of the cell on the proliferation of PEDV, a virus inoculation experiment was performed on a wild type Vero cell and a DBN1-KO-Vero cell. In 24-well plates, 1X 10 inoculations per well 5 Vero cells or DBN1-KO-Vero cells. After 24h incubation, cells were washed with PBS, and PEDV was inoculated at a MOI =0.1 dose, and virus and maintenance solution (high sugar containing 7.5ug/mL pancreatin) were added per wellDMEM complete medium) for a total of 200uL, adsorbed for 2h, and then supplemented with maintenance medium to 500uL per well. Collecting cells 10h and 20h after inoculation for fluorescence quantitative PCR, western blot and IFA detection, collecting cell culture solution for TCID 50 And (6) detecting. The mRNA replication level of PEDV is detected by fluorescent quantitative PCR (polymerase chain reaction), the result is shown in figure 5, the PEDV replication level of the DBN1-KO-Vero cell strain is obviously higher than that of a wild control group 10h and 20h after virus inoculation and the DBN1 gene knockout cell strain DBN1-KO-Vero, and the fact that the PEDV replication is promoted by knockout of the DBN1 gene is indicated; western blot results are shown in FIG. 6, and it is found from the graph that the expression level of the N protein of PEDV is obviously improved 10h and 20h after the DBN1 gene knockout cell strain DBN1-KO-Vero is inoculated; IFA results are shown in FIG. 7, after the DBN1-KO-Vero cells are inoculated with the virus, 10h and 20h are carried out, the fluorescence quantity of the N protein of PEDV is obviously enhanced, and the effect that the DBN1 gene knockout can promote the replication of the PEDV is further verified; TCID 50 As shown in FIG. 8, the titer levels of the DBN1 knock-out cell line DBN1-KO-Vero were significantly increased at 10h and 20h after the inoculation. In particular, the log Titer (TCID) of the virus 20h after PEDV vaccination 50 /ml) is increased from 4.42 of common cells to 4.89 of knockout cells, the titer is improved by nearly 0.5, and the result shows that the DBN1 gene knockout cell strain DBN1-KO-Vero has great potential in PEDV propagation culture.
The mRNA replication level of PEDV is detected by fluorescent quantitative PCR, and the specific operation steps are as follows:
samples RNA was extracted using the Fastpure cell/Tissue toll RNA Isolation Kit V2 Kit and the RNA concentration was measured. And reverse transcribing the RNA to cDNA using HiScript II Q RT Supermix for qPCR reverse transcriptase. The reverse transcription system is as follows: 5 XHiScript II qRT Supermix II 4ul, RNA 1ug, RNase-free ddH 2 O make up the volume to 16uL. The reaction conditions were 55 ℃ for 15min and 85 ℃ for 5s.
The cDNA obtained by reverse transcription was diluted 3 times and subjected to fluorescent quantitative PCR detection using the reagent of ChamQ Universal SYBR qPCR Master Mix. The fluorescent quantitative PCR system is as follows: 2 XChamQ Universal SYBR Qpcr Master Mix10uL, PEDV-F0.4 uL, PEDV-R0.4 uL, cDNA 1uL, RNase-free ddH 2 O is added to the volume of 20uL, and the GAPDH internal reference group is consistent except that the primer is GAPDH-F/R. The reaction conditions are as follows: the pre-denaturation is carried out for 30s at the temperature of 95 ℃,circulating for 40 times at 95 ℃ for 10s and 60 ℃ for 30 s; dissolution curve: 95 ℃ 15s,60 ℃ 60s,95 ℃ 15s. Using relative quantities 2 -△△Ct The method processes and analyzes experimental data, corrects the expression level of the PEDV gene by using an internal reference gene GAPDH, and performs data difference detection according to a t value. The fluorescent quantitative PCR primer sequence is as follows:
PEDV primer sequence:
PEDV-F:5’-CCTCCGTTATAGGACTCGTACTC-3’，
PEDV-R:5’-TTGGAATGATTGGCTTTTCAGA-3’。
GAPDH internal reference primer sequence:
GAPDH-F:5’-TGACCTGCCGTCTGGAAAA-3’，
GAPDH-R:5’-GGAAGAGTGGGTGTCGCTGT-3’。
IFA specific operation steps: washing cells with PBS for 3 times, adding-20 deg.C precooled anhydrous ethanol, and fixing in-20 deg.C refrigerator for 30min; PBS washing 3 times, adding 1; washing with PBS for 3 times, adding FITC goat anti-mouse fluorescent antibody diluted by 1; PBS was washed 3 times, added with 1. PBS was washed 3 times and placed under a fluorescence microscope for observation.
TCID 50 The detection method comprises the following specific operation steps: vero cells were plated in 96-well plates and cultured to grow well over a monolayer. PBS wash 3 times, virus cultures were diluted with a maintenance solution (high-glucose DMEM complete medium containing 7ug/mL pancreatin) in 8 gradients (10) -1 -10 -8 ) 24 replicate wells were set for each gradient, and 100ul of each was added to a 96-well plate, while a blank control well was set containing only maintenance solution. The cells were incubated in a 37 ℃ C.and 5% CO2 cell incubator, during which cytopathic conditions were observed and recorded every day until the cells were not diseased. Counting the number of the pathological cell pores, and calculating the TCID of the virus by a Reed-Muench two-step method 50 。
SEQUENCE LISTING
<110> agricultural science institute of Jiangsu province
<120> method for constructing Vero cell strain with DBN1 gene knocked out and application thereof
<130> 20220114
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Vero cell
<400> 1
<210> 2
<211> 25
<212> DNA
<213> artificial
<220>
<223> sgRNA-DBN1-F
<400> 2
caccgtgcct gagggggctc ctcca 25
<210> 3
<211> 25
<212> DNA
<213> artificial
<220>
<223> sgRNA-DBN1-R
<400> 3
aaactggagg agccccctca ggcac 25
Claims (5)
1. A construction method of a Vero cell strain with a DBN1 gene knocked out is characterized by comprising the following steps:
(1) Finding a target spot of a Vero cell DBN1 gene, and designing corresponding target sgRNA which comprises sgRNA-DBN1-F and sgRNA-DBN1-R;
(2) The PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector is cut by Bbs I endonuclease, and then is connected with a double-stranded molecule formed after sgRNA-DBN1-F and sgRNA-DBN1-R sequences are annealed to obtain a knockout plasmid PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0;
(3) And co-transfecting the knockout plasmid PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0 and lentivirus auxiliary packaging plasmids PSPXA2 and PMD.2G into Vero cells, and screening to obtain a Vero cell strain with the DBN1 gene knocked out.
The sequence of the target point is shown as SEQ ID NO:1 is shown in the specification; the sequences of the sgRNA-DBN1-F and the sgRNA-DBN1-R are shown in SEQ ID NO: 2-3.
2. The method for constructing a Vero cell line with a DBN1 gene knocked out according to claim 1, which is characterized in that puromycin-containing screening is adopted.
3. The use of a Vero cell line with a DBN1 gene knocked out, which is constructed according to claim 1, in the culture of PEDV.
4. The use according to claim 3, wherein the Vero cell line with DBN1 knockout is cultured, PEDV is inoculated, and the culture is continued by using a maintenance solution.
5. The use according to claim 4, characterized in that the maintenance solution is a high-sugar DMEM complete medium containing 7.5ug/mL pancreatin.
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