Application of porcine TAB1 knockout cell line in promotion of PRV proliferation

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to application of a porcine TAB1 knockout cell line in promotion of PRV proliferation. The porcine TAB1 knockout cell line is obtained by transfecting a porcine TAB1 gene knockout plasmid into a cell and then culturing; the porcine TAB1 gene knockout plasmid is obtained by inserting a nucleotide sequence shown as a sequence 1 in a sequence table between a Hind III restriction enzyme cutting site of a Px459M plasmid and a Bbs I restriction enzyme cutting site farthest from the Hind III restriction enzyme cutting site. Compared with a wild type 3D4 / 21 cell line, the porcine TAB1 knockout cell line obviously promotes PRV virus proliferation, can be used for PRV proliferation research, and is further applied to preparation of PRV vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and more specifically, relates to the application of a porcine TAB1 knockout cell line in promoting the proliferation of PRV. Background Art

[0002] Pseudorabies virus (PRV) is an enveloped, linear, double-stranded DNA virus with a genome size of 150 kb. It can encode more than 70 proteins and belongs to the genus Varicellavirus of the subfamily Alphaherpesvirinae of the family Herpesviridae. PRV caused by PRV infection is one of the major diseases that seriously affects the pig industry. The main symptoms are abortion and stillbirth in pregnant sows, infertility in boars, mass death of newborn piglets, breathing difficulties and growth stagnation in fattening pigs, etc. It is listed as a Class B infectious disease by the World Organization for Animal Health (OIE). Although the natural host of PRV is pigs, it can also infect other animals such as cattle, sheep, cats and dogs. The latest research shows that PRV can directly infect humans, causing serious damage to the nervous system and respiratory system, indicating that PRV is a zoonotic virus that can infect humans under certain conditions. Given that PRV infection has adverse effects on the breeding industry and human health, studying how to prevent and treat PRV is of great significance to animal disease prevention and control and human health protection.

[0003] Transforming growth factor β-activated kinase 1 (TAK1) is a member of the MAPK (MAPKkinase kinase, MAPKKK) kinase family and is involved in the regulation of various physiological and pathological processes, especially the activation of NF-κB and MAPK signaling pathways mediated by Toll like receptors (TLRs). TAK1-binding protein 1 (TAB1) is a protein that interacts with TAK1. It binds to TAK1 through the C-terminal domain to form a TAB1 / TAK1 / TAB2 / TAB3 complex, which is an important node protein in the NF-κB signaling pathway. This complex can further activate the IKK complex (IKKα / IKKβ / IKKγ [NEMO]), thereby activating the NF-κB signaling pathway, initiating the production of type I IFN, and exerting an antiviral effect. Whether porcine TAB1 (sTAB1) protein can inhibit the replication and proliferation of PRV has not been reported.

[0004] CRISPR / Cas9 gene editing technology consists of Cas9 nuclease and specific guide RNA, which can achieve a variety of gene editing such as knockout, knock-in and site-directed mutagenesis of target gene sites. It is crucial to develop a method to knock out the sTAB1 gene from the pig cell genome, which can lay the foundation for the study of the antiviral mechanism of sTAB1 and the development of PRV vaccines. Summary of the invention

[0005] The present invention provides application of a porcine TAB1 knockout cell line in promoting the proliferation of PRV.

[0006] Combining the existing research progress of TAB1 in innate immunity, using CRISPR / Cas9 gene editing technology, a porcine TAB1 (sTAB1) gene knockout plasmid Px459M-sTAB1-KO and cell line sTAB1-KO-3D4 / 21 were provided to lay the foundation for the study of the antiviral mechanism of sTAB1.

[0007] The present invention also provides a construction method of the sTAB1 gene knockout plasmid Px459M-sTAB1-KO and the cell line sTAB1-KO-3D4 / 21.

[0008] The present invention also provides the use of the sTAB1 gene knockout cell line sTAB1-KO-3D4 / 21 in promoting the proliferation of PRV.

[0009] The present invention is obtained by following steps:

[0010] Application of a porcine TAB1 knockout cell line in promoting the proliferation of PRV, wherein the porcine TAB1 knockout cell line is obtained by transfecting cells with a porcine TAB1 gene knockout plasmid and then culturing the cells;

[0011] The porcine TAB1 gene knockout plasmid is obtained by inserting the nucleotide sequence shown in sequence 1 in the sequence list between the Hind III restriction site of the Px459M plasmid and the Bbs I restriction site farthest from the Hind III restriction site.

[0012] The application preferably promotes PRV proliferation, specifically increasing the PRV virus copy number, increasing the virus titer, and increasing the expression level of serum albumin viral glycoprotein gB.

[0013] The application is preferably used to promote the proliferation of PRV, wherein the PRV is the PRV Bartha-K61 vaccine strain.

[0014] The application, preferably the porcine TAB1 gene knockout plasmid, is obtained by the following steps:

[0015] (1) Design two pairs of sTAB1 guide RNA primers, whose sequences are as follows:

[0016] sTAB1-KO-2F:5'-CACCGCTTGTAATCCCCAATCCGCC-3'

[0017] sTAB1-KO-2R:5'-AAACGGCGGATTGGGGATTACAAG-3'

[0018] sTAB1-KO-5F:5'-CACCGTCGAGTACGGCACGGACACG-3'

[0019] sTAB1-KO-5R:5'-AAACCGTGTCCGTGCCGTACTCGA-3'

[0020] The two pairs of guide RNA primers are phosphorylated and annealed to obtain sticky-end double-stranded sTAB1-KO-2 and sTAB1-KO-5;

[0021] (2) Construction of sTAB1 gene knockout plasmid Px459M-sTAB1-KO

[0022] The Px459M vector and the EZ-Guide-XH vector were digested with Bbs I and recovered by gel lysis. Then, two pairs of sTAB1 guide RNA primers were connected to the digested Px459M vector and the EZ-Guide-XH vector, respectively, to obtain the recombinant plasmids Px459M-sgRNA2 and EZ-Guide-XH-sgRNA5.

[0023] The recombinant plasmids Px459M-sgRNA2 and EZ-Guide-XH-sgRNA5 were double-digested with Hind III / Xho I to obtain the vector fragment Px459M-sTAB1-KO-2 and the gene fragment sTAB1-KO-5, respectively. The vector fragment and the gene fragment were connected to obtain the porcine TAB1 gene knockout plasmid Px459M-sTAB1-KO.

[0024] For the application, the preferred phosphorylation and annealing conditions are as follows:

[0025] Reaction system: sTAB1-KO-2F / sTAB1-KO-5F 1μL, concentration is 100μM, sTAB1-KO-2R / sTAB1-KO-5R 1μL, concentration is 100μM, 10×T4 PNK Buffer 1μL, T4 PNK 1μL, ddH2O 6μL; reaction conditions are: 37℃ 30min, 95℃ 5min, cooling to 25℃ at a gradient of -0.1℃ / s, 25℃ 5min, 4℃ 5min.

[0026] In the application, 447 bases in the coding region of the pig TAB1 gene in the pig TAB1 knockout cell line are deleted, and the retained base sequence is shown in sequence 2 in the sequence table.

[0027] Beneficial effects of the present invention:

[0028] (1) The porcine TAB1 knockout cell line sTAB1-KO-3D4 / 21 constructed by the present invention does not express sTAB1 protein, and can be applied to the study of the antiviral effect of sTAB1;

[0029] (2) PRV infection experiments have shown that the porcine TAB1 knockout cell line sTAB1-KO-3D4 / 21 cell line significantly promotes PRV virus proliferation compared with the wild-type 3D4 / 21 cell line. The knockout cell line can be used for the study of PRV proliferation and then for the preparation of PRV vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A is the PCR identification of plasmid Px459M-sgRNA2, where M is DL2000 DNA Marker and 1-2 are the PCR amplification results of Px459M-sgRNA2; B is the PCR identification of plasmid EZ-Guide-XH-sgRNA5, where M is DL2000 DNA Marker and 1-2 are the PCR amplification results of EZ-Guide-XH-sgRNA5; C is the Hind III / Xho I double enzyme digestion identification of plasmid Px459M-sTAB1-KO, where M is DL10000 DNA Marker and 1-2 are the Hind III / Xho I double enzyme digestion results of Px459M-sTAB1-KO plasmid.

[0031] Figure 2 A is the RT-PCR amplification result of sTAB1 gene in sTAB1-KO-3D4 / 21 and 3D4 / 21 cells, where M is DL2000 DNA Marker, 1 is the RT-PCR amplification result of sTAB1-KO-3D4 / 21, and 2 is the RT-PCR amplification result of sTAB1 gene in 3D4 / 21 cells; B is the sequence alignment of RT-PCR products of sTAB1 gene in sTAB1-KO-3D4 / 21 and 3D4 / 21 cells; C is the protein expression of sTAB1 in sTAB1-KO-3D4 / 21 and 3D4 / 21 cells.

[0032] Figure 3 A is the absolute fluorescence quantitative detection of the viral copy number of PRV Bartha-K61 vaccine strain in 3D4 / 21 cells and sTAB1-KO-3D4 / 21 cells; B is TCID 50The virus titer of PRV Bartha-K61 vaccine strain in 3D4 / 21 cells and sTAB1-KO-3D4 / 21 cells was detected; C is the expression of PRV Bartha-K61 vaccine strain gB protein in 3D4 / 21 (lanes 1,3,5and 7) cells and sTAB1-KO-3D4 / 21 cells (lanes 2,4,6and 8) detected by Western blotting; D is the grayscale analysis of the relative expression level of PRV Bartha-K61 vaccine strain gB protein in the two cells in Figure C. DETAILED DESCRIPTION

[0033] The technical solution of the present application is described in detail below in conjunction with specific embodiments.

[0034] Example 1: Construction and identification of sTAB1 gene knockout plasmid Px459M-sTAB1-KO

[0035] 1.1 Design and synthesis of TAB1 guide RNA primer

[0036] Two pairs of specific guide RNA primers were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd. The sequences are as follows:

[0037] sTAB1-KO-2F:5'-CACCGCTTGTAATCCCCAATCCGCC-3'

[0038] sTAB1-KO-2R:5'-AAACGGCGGATTGGGGATTACAAG-3'

[0039] sTAB1-KO-5F:5'-CACCGTCGAGTACGGCACGGACACG-3'

[0040] sTAB1-KO-5R:5'-AAACCGTGTCCGTGCCGTACTCGA-3'

[0041] 1.2 Primer phosphorylation and annealing

[0042] The two pairs of guide RNA primers synthesized above were diluted to 100 μM, phosphorylated and annealed to form sticky end double strands to obtain TAB1-KO-2 and TAB1-KO-5. The reaction system is: TAB1-KO-2F / TAB1-KO-5F 1 μL, TAB1-KO-2R / TAB1-KO-5R 1 μL, 10×T4 PNK Buffer 1 μL, T4 PNK 1 μL, ddH2O 6 μL. The reaction conditions are: 37℃30min, 95℃5min, cooling to 25℃, 25℃5min, 4℃5min according to the gradient of -0.1℃ / s. After the system is prepared, first use a centrifuge to mix it, then place it in a PCR instrument for amplification reaction, and the reaction product is placed at -20℃ for use.

[0043] 1.3 Construction and identification of knockout plasmid Px459M-sTAB1-KO

[0044] The Px459M and EZ-Guide-XH vectors were digested with Bbs I at 37°C for 2h, and the products were subjected to 1% agarose gel electrophoresis. After the target band was cut from the gel, gel recovery was performed according to the instructions of the DNA purification and recovery kit. The two pairs of guide RNAs were connected to the Px459M vector and the EZ-Guide-XH vector by T4DNALigase to construct plasmids Px459M-sgRNA2 and EZ-Guide-XH-sgRNA5, and transformed with DH5α competent cells, spread on LB solid culture medium plates containing ampicillin resistance, inverted plates, and cultured at 37°C overnight; the next day, single colonies were picked and placed in LB liquid culture medium containing ampicillin resistance, and cultured at 37°C 200r / min overnight with shaking;

[0045] The next day, plasmids were extracted and identified by PCR. Px459M-sgRNA2 plasmid was amplified with TAB1-KO-2F and CAG-R primers; EZ-Guide-XH-sgRNA5 plasmid was amplified with TAB1-KO-5F and M13F primers.

[0046] The PCR primers used were

[0047] CAG-R:5'-GTACTGGGCACAATGCCAG-3'

[0048] M13F:5'-TGTAAAACGACGGCCAGT-3'

[0049] The PCR amplification results are shown in Figure 1In Figures A and B, the plasmids identified as positive were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing using CAG-R and M13F, respectively. The correctly sequenced plasmids were named Px459M-sgRNA2 and EZ-Guide-XH-sgRNA5, respectively.

[0050] The plasmids Px459M-sgRNA2 and EZ-Guide-XH-sgRNA5 were double-digested with Hind III / Xho I, and the vector fragment Px459M-sTAB1-KO-2 and the gene fragment sTAB1-KO-5 were recovered by gel recovery. The vector fragment and the gene fragment were connected overnight at 16°C and transformed with DH5α competent cells. The plasmids were extracted from the bacterial solution and identified by Hind III / Xho I double-digestion. The results are as follows Figure 1 As shown in C, the positively identified plasmid was sent to Beijing Qingke Biotechnology Co., Ltd. for CAG-R sequencing. The recombinant plasmid with correct identification and gene sequence alignment was named Px459M-sTAB1-KO, and the nucleotide sequence shown in sequence 1 in the sequence table was inserted between the Hind III restriction site of the Px459M plasmid and the Bbs I restriction site farthest from the Hind III restriction site.

[0051] Example 2: Construction and identification of sTAB1 gene knockout 3D4 / 21 cell line

[0052] 2.1 Construction of sTAB1 gene knockout 3D4 / 21 cell line

[0053] 3D4 / 21 cells were cultured in RPMI 1640 cell culture medium containing a mixture of 10% FBS and 1% penicillin-streptomycin, and the incubator was set at 37°C and 5% CO2 concentration. When the cells were full and in good growth condition, the 3D4 / 21 cells were digested and passaged, and 12-well plates were plated with 1 mL of cells per well, and cultured overnight. When the cell density reached 80% to 90%, the 3D4 / 21 cells were tested for puromycin tolerance, and the concentrations were set to: 0, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 μg / mL. When the 3D4 / 21 cells to which puromycin of various concentrations were added were cultured to the 3rd day, the lowest concentration of puromycin that could cause all cell death was screened out as the optimal drug concentration for the 3D4 / 21 cell knockout cell line. The optimal puromycin concentration screened by the present invention was 2.0 μg / mL.

[0054] 3D4 / 21 cells were plated in 12-well plates, and non-transfected cells were set as negative controls. When the cell density reached 80% to 90%, 3000 instructions, Px459M-sTAB1-KO plasmid was transfected into 3D4 / 21 cells. After transfection for 24 hours, the supernatant was discarded, and the culture medium containing puromycin was added to continue culturing for 3d to 4d. All the control cells that were not transfected with the plasmid died, while the cells transfected with the knockout plasmid had live resistant clones. The supernatant was discarded, and the culture medium without puromycin was replaced to continue culturing. The cells were diluted by limiting dilution method, and 1 to 2 cells were cultured in each well of a 96-well plate. The growth status of monoclonal cells in each well was observed. The cells in the 96-well plate were expanded to 48-well plates according to the growth status. After full growth, the cells in the 48-well plate were expanded to 24-well plates for culture. During the culture process, some cells were taken for identification, and the remaining cells continued to be cultured.

[0055] 2.2 RT-PCR identification

[0056] The cells taken out in the above process and wild-type 3D4 / 21 cells were respectively used for total RNA extraction using RNA-easy Isolation Reagent reagent, and used as templates, and RT-PCR amplification was performed using primers sTAB1-F and sTAB1-R for amplifying the sTAB1 gene sequence according to the instructions of HiScript II One Step RT-PCR Kit (DyePlus). The reaction system was: One Step Enzyme Mix 2.5μL, 2×One Step Mix 25μL, sTAB1-F 2μL, sTAB1-R 2μL, RNA 1μg, and RNase Free ddH2O was added to 50μL. The reaction conditions were 50℃ 30min; 94℃ 3min; 94℃ 30s, 63℃ 30s, 72℃ 2min, 35 cycles; 72℃ 7min. After the system was prepared, it was first mixed by centrifuge, and then placed in a PCR instrument for amplification reaction. The reaction product was stored at 4℃.

[0057] The RT-PCR primers used were

[0058] sTAB1-F:5'-ATGGCGGCGCAGAGG-3'

[0059] sTAB1-R:5'-CTACGGCGCCGTCGC-3'

[0060] The RT-PCR amplification products of positive clones and control cells were subjected to 1% agarose gel electrophoresis. Figure 2As shown in A. The size of the electrophoresis band of the screened sTAB1-KO-3D4 / 21 cells is about 1068 bp, and the size of the electrophoresis band of the control group 3D4 / 21 cells is about 1515 bp. The band of sTAB1-KO-3D4 / 21 cells around 1068 bp was recovered by gel and sent for sequencing. The results are shown in Figure 2 As shown in B, a total of 447 nucleotides of the sTAB1 gene of the knockout cells were knocked out, and the retained nucleotide sequence is shown in Sequence 2. The knockout cell line was named sTAB1-KO-3D4 / 21.

[0061] 2.3 Western blotting

[0062] The sTAB1-KO-3D4 / 21 cells and wild-type 3D4 / 21 cells taken out in the above process were inoculated in 12-well plates, and the cells were collected for total protein extraction after the cells were fully grown. The supernatant was discarded, and the cell samples were washed twice with PBS precooled at 4°C. The 12-well plate was placed on ice, and an appropriate amount of cell protein lysis buffer was added for lysis. At the same time, the lysis buffer was added with protease inhibitor PMSF (1:100) and fully mixed. Lysis was performed on ice for 15 minutes, and the plate was shaken several times every 5 minutes to allow the cells to be more fully lysed. The cells blown off by the gun tip were collected into a 1.5mL EP tube, placed in a 4°C centrifuge at 12000r / min for 10 minutes, and the supernatant was taken into a new precooled 1.5mL EP tube, and an appropriate amount of loading buffer was added. After mixing, it was boiled in boiling water for 15 minutes to denature the protein. The protein sample was used to identify the protein expression of TAB1. The samples were subjected to 12% SDS-PAGE gel electrophoresis with a starting voltage of 90V. When the protein bands ran to the separation gel, the voltage was adjusted to 120V. The proteins were transferred to the NC membrane using wet transfer at 100V for 70min. The membranes were blocked with 5% skim milk powder at room temperature for 2h. TAB1 antibody and internal reference β-actin antibody diluted in primary antibody diluent were used as primary antibodies, incubated overnight at 4°C, and washed 5 times with TBST for 5min each time. HRP-labeled goat anti-rabbit IgG was used as secondary antibody, incubated at room temperature for 1h, and washed 5 times with TBST for 5min each time. The developer was prepared according to the instructions of the Sparkjade ECL super ultrasensitive chemiluminescence kit for protein color development, and the images were collected and analyzed by Bio-RAD gel imager.

[0063] Western blotting was used to detect the expression of sTAB1 protein in 3D4 / 21 and sTAB1-KO-3D4 / 21 cells. The results are shown in Figure 2 As shown in center C, sTAB1 protein expression was detected in 3D4 / 21 cells, but not in sTAB1-KO-3D4 / 21 cells.

[0064] Example 3: Use of sTAB1 knockout cell line in the proliferation of PRV Bartha-K61 vaccine strain

[0065] 3.1 Effect of sTAB1 knockout on viral copy number

[0066] 3D4 / 21 cells and sTAB1-KO-3D4 / 21 cells were inoculated into 12-well plates, respectively. When the cell density reached 90%, the PRV strain was diluted to MOI = 0.05 using RPMI 1640 cell culture medium containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin. 1 mL was added to each well of the 12-well plate and the plate was placed in a cell culture incubator at 37°C and 5% CO2 and cultured for 6 h, 12 h, 18 h, and 24 h. The cell supernatant was collected for PRV copy number and titer detection, and the cells were collected for PRV gB protein level detection.

[0067] According to the instructions of Simply P Virus DNA / RNA Extraction Kit, the total DNA from the PRV strain was extracted, and the PRV-gD gene fragment was amplified using the DNA as a template with PRV-gD-F / PRV-gD-R primers. The obtained PCR product was purified and used as a standard for the PRV-gD fragment. The viral copy number was calculated based on the concentration of the PRV-gD fragment, and the concentration was adjusted and diluted 10 times with ddH2O. The concentration after dilution was 10 10 ~10 3 copies / μL. DNA was extracted from the cell supernatant sample and used as a template. Together with the gradiently diluted PRV-gD positive standard, PRV-gD-F / PRV-gD-R primers were used to detect the PRV copy number by absolute fluorescence quantitative PCR according to the instructions of the 2×RealStarGreen Fast Mixture kit. Each group of samples was repeated 3 times in parallel. The reaction system is: 2×RealStar Green Fast Mixture 10μL, PRV-gD-F 1μL, PRV-gD-R1μL, standard / DNA2μL, ddH2O 6μL. After the system is prepared, mix it well and then centrifuge it instantly, and place it in a PCR instrument for amplification reaction. The reaction conditions are 95℃1s; 95℃5s, 55℃20s, 72℃20s, 40 cycles; 65℃15s. The PRV copy number in each sample is obtained by comparing the Ct value drawn with the standard curve. The results are shown in the following figure. Figure 3 As shown in A, at 6h, 12h, 18h, and 24h after virus infection, the copy numbers of PRV Bartha-K61 strain in sTAB1-KO-3D4 / 21 cells were significantly higher than those in 3D4 / 21 cells (P<0.05).

[0068] The fluorescent quantitative PCR primers used were

[0069] PRV-gD-F:5'-TGAACATCCTCACCGACTTC-3'

[0070] PRV-gD-R:5'-TAGAACGGCGTCAGGAATC-3'

[0071] 3.2 Effect of sTAB1 knockout on viral titer

[0072] PK-15 cells were cultured in a high-glucose DMEM cell culture medium containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin. PK-15 cells were inoculated in a 96-well plate and cultured in a cell culture incubator at 37°C and 5% CO2. When the cell density reached 90%, a high-glucose DMEM cell culture medium containing a mixture of 2% fetal bovine serum and 1% penicillin-streptomycin was used to culture the supernatant samples of cells infected with the PRV strain in 3.1 for 6h, 12h, 18h, and 24h for 10 -1 ~10 -10 Gradient dilution, 4 replicates per gradient, 100 μL per well. Aspirate the original cell supernatant in the 96-well plate, add the diluted virus solution, and set up an empty cell control without virus solution. Continue to observe for 72 hours to count the cytopathic effect, and calculate TCID according to the Reed-Muench method. 50 , the results are as follows Figure 3 As shown in B, at 6h, 12h, 18h, and 24h after virus infection, the PRV Bartha-K61 virus titer in sTAB1-KO-3D4 / 21 cells was significantly higher than that in 3D4 / 21 cells (P<0.05).

[0073] 3.3 Effect of sTAB1 knockout on viral gB protein

[0074] Take the cell samples infected with PRV Bartha-K61 strain in 3.1 for 6h, 12h, 18h, and 24h, and refer to the Western blotting method in step 2.3 to collect cells and extract proteins. Perform SDS-PAGE gel electrophoresis, use diluted PRV gB antibody and internal reference β-actin antibody as primary antibodies, and HRP-labeled goat anti-mouse IgG and goat anti-rabbit IgG as secondary antibodies to detect the expression level of PRV gB protein. Use Image J software to perform grayscale analysis of protein bands. The results are shown in Figure 2. Figure 3 As shown in C and D, at 6h, 12h, 18h, and 24h after virus infection, the expression levels of PRV gB protein in sTAB1-KO-3D4 / 21 cells were significantly higher than those in 3D4 / 21 cells (P<0.05).

[0075] Primary porcine alveolar macrophages cannot be passaged, while the 3D4 / 21 cell line is transformed from primary porcine alveolar macrophages. It is a porcine alveolar macrophage that can be passaged and is a commercially stable and commonly used porcine immune cell. Therefore, this application chooses to construct a sTAB1 knockout cell line in 3D4 / 21 cells. The knockout cell line described in the applicant's published paper (Xia et al., 2024) was constructed in the PAM-KNU cell line, which is an immortalized porcine alveolar macrophage cell line transfected with human telomerase reverse transcriptase (hTERT) and is more suitable for PRRSV-related research. The two cell lines have their own advantages and disadvantages when proliferating multiple viruses, and there is no value in reference to each other.

[0076] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited by the embodiments. Any other changes, modifications, combinations, substitutions and simplifications made without departing from the spirit and principles of the present invention shall be considered as equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. Application of a porcine TAB1 knockout cell line in promoting the proliferation of PRV, wherein the porcine TAB1 knockout cell line is obtained by transfecting cells with a porcine TAB1 gene knockout plasmid and then culturing the cells; The porcine TAB1 gene knockout plasmid is obtained by inserting the nucleotide sequence shown in sequence 1 in the sequence list between the Hind III restriction site of the Px459M plasmid and the Bbs I restriction site farthest from the Hind III restriction site.

2. The use according to claim 1, characterized in that Promote PRV proliferation, specifically by increasing the PRV virus copy number, increasing the virus titer, and increasing the expression of viral glycoprotein gB.

3. The use according to claim 1, characterized in that In promoting the proliferation of PRV, the PRV is the PRV Bartha-K61 vaccine strain.

4. The use according to claim 1, characterized in that The porcine TAB1 gene knockout plasmid is obtained by the following steps: (1) Design two pairs of sTAB1 guide RNA primers, whose sequences are as follows: sTAB1-KO-2F:5'-CACCGCTTGTAATCCCCAATCCGCC-3' sTAB1-KO-2R:5'-AAACGGCGGATTGGGGATTACAAG-3' sTAB1-KO-5F:5'-CACCGTCGAGTACGGCACGGACACG-3' sTAB1-KO-5R:5'-AAACCGTGTCCGTGCCGTACTCGA-3' The two pairs of guide RNA primers are phosphorylated and annealed to obtain sticky-end double-stranded sTAB1-KO-2 and sTAB1-KO-5; (2) Construction of sTAB1 gene knockout plasmid Px459M-sTAB1-KO The Px459M vector and the EZ-Guide-XH vector were digested with Bbs I and recovered by gel lysis. Then, two pairs of sTAB1 guide RNA primers were connected to the digested Px459M vector and the EZ-Guide-XH vector, respectively, to obtain the recombinant plasmids Px459M-sgRNA2 and EZ-Guide-XH-sgRNA5. The recombinant plasmids Px459M-sgRNA2 and EZ-Guide-XH-sgRNA5 were double-digested with Hind III / Xho I to obtain the vector fragment Px459M-sTAB1-KO-2 and the gene fragment sTAB1-KO-5, respectively. The vector fragment and the gene fragment were connected to obtain the porcine TAB1 gene knockout plasmid Px459M-sTAB1-KO.

5. The use according to claim 4, characterized in that Phosphorylation and annealing conditions were as follows: Reaction system: sTAB1-KO-2F / sTAB1-KO-5F 1μL, concentration is 100μM, sTAB1-KO-2R / sTAB1-KO-5R 1μL, concentration is 100μM, 10×T4 PNK Buffer 1μL, T4 PNK 1μL, ddH2O 6μL; reaction conditions are: 37℃ 30min, 95℃ 5min, cooling to 25℃ at a gradient of -0.1℃ / s, 25℃ 5min, 4℃ 5min.

6. The use according to claim 4, characterized in that In the porcine TAB1 knockout cell line, 447 bases are deleted in the coding region of the porcine TAB1 gene, and the retained base sequence is shown in Sequence 2 in the sequence listing.

Images