Pig trim56 knockout plasmid, cell line and construction method and application thereof

Abstract

The present application relates to the technical field of molecular biology, in particular to the construction of a pig TRIM56 gene knockout plasmid, the construction of a pig TRIM56 knockout PAM-KNU cell line, and the fact that the knockout cell line can significantly promote the proliferation of PRRSV and can be used for antiviral research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and more particularly relates to a pig TRIM56 knockout plasmid, a cell line and a construction method and application thereof. BACKGROUND

[0002] TRIM56 belongs to the C-V subfamily of the Tripartite motif (TRIM) family, and contains three conserved domains of RING, B-box and coiled-coil at the N-terminus, but the C-terminal structure is not very clear. The pig TRIM56 protein is composed of 755 amino acids. TRIM56 has a ring-dependent E3 ubiquitin ligase activity and self-binding in cells, and has the ability to mediate ubiquitin transfer to heterologous substrates and itself. TRIM56 can activate the TLR3-mediated antiviral signaling pathway, and can inhibit the proliferation of type I human immunodeficiency virus, bovine viral diarrhea virus, coronavirus, etc. The antiviral activity of TRIM56 depends on the activity of its E3 ligase and the integrity of its C-terminal region.

[0003] Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the Arterivirus of the Arteriviridae family, and is a single-stranded positive-sense RNA virus with a capsule. PRRSV can cause porcine reproductive and respiratory syndrome (PRRS) after infecting pigs, which is a highly contagious disease. Its characteristic manifestations are abortion, stillbirth and production of mummies in pregnant sows, respiratory disorders in piglets, and destruction of the pig immune system, causing mixed or secondary infections. Since the outbreak of PRRSV, it has caused serious economic losses to the global pig industry, and the World Organization for Animal Health (OIE) has classified it as a class B infectious disease. At present, China mainly uses attenuated live vaccine immunization to prevent and control PRRS, but the production process needs to be improved and improved, and exploring cell tools to promote the proliferation of PRRSV can provide new ideas and methods for the prevention and control of the disease.

[0004] Whether pig TRIM56 (sTRIM56) inhibits PRRSV replication and proliferation, no relevant research reports are seen. The CRISPR / Cas9 gene editing technology is composed of a Cas9 nuclease and a specific guide RNA, and can realize a variety of gene editing such as target gene site knockout, knock-in and site-directed mutation. The PAM-KNU cell line is a human telomerase reverse transcriptase (hTERT) immortalized pig alveolar macrophage cell line, and has the characteristics of immune cells and passable characteristics, and is an ideal pig cell line tool for studying innate immunity. It is crucial to develop a method for knocking out the sTRIM56 gene from the pig cell genome, which can lay a foundation for sTRIM56 antiviral mechanism research and PRRS vaccine development. SUMMARY

[0005] The purpose of the present application is to combine the research progress of existing TRIM56 in innate immunity, to provide a pig TRIM56 gene knockout plasmid Px459M-sTRIM56-KO and cell line sTRIM56-KO-PAM-KNU by using CRISPR / Cas9 gene editing technology, and to lay a foundation for pig TRIM56 antiviral mechanism research.

[0006] The present application also provides a construction method of the pig TRIM56 gene knockout plasmid Px459M-sTRIM56-KO and cell line sTRIM56-KO-PAM-KNU.

[0007] The present application also provides an application of the pig TRIM56 gene knockout cell line sTRIM56-KO-PAM-KNU in proliferating PRRSV R98 vaccine strain.

[0008] The present application is obtained by the following steps:

[0009] A pig TRIM56 gene knockout plasmid, the nucleotide sequence inserted between the Hind III enzyme cutting site of the Px459M plasmid and the Bbs I enzyme cutting site farthest from the Hind III enzyme cutting site is the nucleotide sequence of sequence 1 in the sequence table.

[0010] A preparation method of a pig TRIM56 gene knockout plasmid, characterized by being obtained by the following steps:

[0011] (1) Design two pairs of pig TRIM56 guide RNA primers, and the sequences are as follows:

[0012] TRIM56-KO-5F: 5'-GGCGGTCGCCTTTGCCCGTAGTTT-3'

[0013] TRIM56-KO-5R: 5'-AAACTACGGGCAAAGGCGACCGCC-3'

[0014] TRIM56-KO-7F: 5'-GTCGTGATCCTCGATCCCAAGTTT-3'

[0015] TRIM56-KO-7R: 5'-AAACTTGGGATCGAGGATCACGAC-3'

[0016] By primer phosphorylation and annealing, sticky end double-stranded TRIM56-KO-5, TRIM56-KO-7 is formed.

[0017] (2) Construction of pig TRIM56 gene knockout plasmid Px459M-sTRIM56-KO

[0018] The Px459M vector and the EZ-Guide-XH vector are cut by Bbs I enzyme and gel recovered, and two pairs of guide RNA are inserted into the Px459M vector and the EZ-Guide-XH vector to construct plasmids Px459M-sgRNA5 and EZ-Guide-XH-sgRNA7, which are identified by PCR and sequencing analysis. The results show that the recombinant plasmids Px459M-sgRNA5 and EZ-Guide-XH-sgRNA7 are successfully constructed.

[0019] The plasmids Px459M-sgRNA5 and EZ-Guide-XH-sgRNA7 are double digested with Hind III / Xho I to obtain the vector fragment Px459M-sTRIM56-KO-5 and the gene fragment sTRIM56-KO-7, and then the vector fragment and the gene fragment are connected and transformed, and the plasmid is extracted and identified by Hind III / Xho I double digestion and sequencing analysis. The results show that the knockout plasmid Px459M-sTRIM56-KO is successfully constructed.

[0020] The preparation method optimizes the phosphorylation and annealing conditions as follows:

[0021] The reaction system is: TRIM56-KO-5F / TRIM56-KO-7F 1 μL, TRIM56-KO-5R / TRIM56-KO-7R 1 μL, 10×T4 PNK Buffer 1 μL, T4 PNK 1 μL, ddH2O 6 μL. The reaction conditions are: 37℃ for 30 min, 95℃ for 5 min, and cooling to 25℃ at a gradient of-0.1℃ / s, 25℃ for 5 min, and 4℃ for 5 min.

[0022] A pig TRIM56 gene knockout cell line, a Px459M-sTRIM56-KO plasmid is transfected into PAM-KNU cells, cells are screened by puromycin, cells are diluted by limited dilution method, and the obtained single clone cells are sTRIM56-KO-PAM-KNU cell lines.

[0023] The application, the sTRIM56 knockout cell line promotes the proliferation of PRRSV in host cells.

[0024] The beneficial effects of the present application are:

[0025] (1) The sTRIM56 protein in the sTRIM56-KO-PAM-KNU cell line constructed by the present application is not expressed, and can be applied to the research of sTRIM56 antiviral effect;

[0026] (2) It is proved by PRRSV infection experiment that the sTRIM56-KO-PAM-KNU cell strain significantly promotes the proliferation of PRRSV R98 strain compared with the wild type PAM-KNU cell strain, and the knockout cell line can be used for the research of PRRSV proliferation. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A is the PCR identification of the Px459M-sgRNA5 plasmid, wherein M is DL2000 DNA Marker, and 1-2 is the PCR amplification result of the Px459M-sgRNA5; B is the PCR identification of the EZ-Guide-XH-sgRNA7 plasmid, wherein M is DL2000 DNA Marker, and 1-2 is the PCR amplification result of the EZ-Guide-XH-sgRNA7; C is the Hind III / Xho I double enzyme digestion identification of the Px459M-sTRIM56-KO plasmid, wherein M is DL10000 DNA Marker, and 1-2 is the Hind III / Xho I double enzyme digestion result of the Px459M-sTRIM56-KO plasmid;

[0028] Figure 2 A is the RT-PCR amplification result of the sTRIM56 gene of the sTRIM56-KO-PAM-KNU and PAM-KNU cells, wherein M is DL5000 DNA Marker; B is the sequence alignment diagram of the RT-PCR product of the sTRIM56 gene of the sTRIM56-KO-PAM-KNU and PAM-KNU cells; C is the Western blotting detection of the sTRIM56 protein expression in the sTRIM56-KO-PAM-KNU and PAM-KNU cells;

[0029] Figure 3A is absolute fluorescent quantitative detection of PRRSV R98 strain virus copy number in PAM-KNU and sTRIM56-KO-PAM-KNU cells; B is TCID 50 A is absolute fluorescent quantitative detection of PRRSV R98 strain virus copy number in PAM-KNU and sTRIM56-KO-PAM-KNU cells; B is TCID DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be specifically described below in combination with specific examples.

[0031] Example 1: Construction and identification of pig TRIM56 gene knockout plasmid Px459M-sTRIM56-KO

[0032] 1.1 Design and synthesis of primers

[0033] Two pairs of specific guide RNA primers were designed by CRISPR guide RNA online design tool (http: / / www.addgene.org / CRISPR / ) combined with the exon region of pig TRIM56 (GenBank No. KJ881160) genome sequence published in NCBI database, and the primers were synthesized by Beijing Qikexin Biotechnology Co., Ltd., and the sequences were as follows:

[0034] TRIM56-KO-5F: 5'-GGCGGTCGCCTTTGCCCGTAGTTT-3'

[0035] TRIM56-KO-5R: 5'-AAACTACGGGCAAAGGCGACCGCC-3'

[0036] TRIM56-KO-7F: 5'-GTCGTGATCCTCGATCCCAAGTTT-3'

[0037] TRIM56-KO-7R: 5'-AAACTTGGGATCGAGGATCACGAC-3'

[0038] 1.2 Phosphorylation and annealing of primers

[0039] The primers of the guide RNA synthesized above were diluted to 100 μM, and two pairs of primers were phosphorylated and annealed to form sticky-ended double-stranded to obtain TRIM56-KO-5 and TRIM56-KO-7, and the reaction system was as follows: TRIM56-KO-5F / TRIM56-KO-7F 1 μL, TRIM56-KO-5R / TRIM56-KO-7R 1 μL, 10×T4 PNK Buffer 1 μL, T4 PNK 1 μL, ddH2O 6 μL. After the system was prepared, it was mixed and placed in a PCR instrument for amplification reaction, and the reaction conditions were as follows: 37 °C for 30 min, 95 °C for 5 min, -0.1 °C / s gradient cooling to 25 °C, 25 °C for 5 min, and 4 °C for 5 min. The reaction product was placed at -20 °C for standby.

[0040] 1.3 Construction and identification of gene knockout plasmid

[0041] Px459M and EZ-Guide-XH vectors were digested with Bbs I at 37 °C for 2 h, and the product was subjected to 1% agarose gel electrophoresis, and the gel was recovered using a DNA purification recovery kit. The two pairs of guide RNAs were connected to the Px459M vector and the EZ-Guide-XH vector, respectively, by T4 DNA Ligase, and the DH5α competent cells were transformed. The culture was incubated at 37 °C and 200 r / min overnight. The next day, single colonies were picked into LB medium containing ampicillin, and incubated at 37 °C and 200 r / min overnight. The next day, the plasmid was extracted and subjected to PCR identification. The sgRNA5 plasmid inserted into Px459M was amplified using TRIM56-KO-5F and CAG-R primers (5'-GTACTGGGCACAATGCCAG-3'), and the sgRNA7 plasmid inserted into EZ-Guide-XH was amplified using TRIM56-KO-7F and M13F primers (5'-TGTAAAACGACGGCCAGT-3'), and the results are shown in Figs. A and B. Figure 1 The plasmids identified as positive were sent to Beijing Qikang Biotechnology Co., Ltd. for sequencing using CAG-R and M13F, respectively. The plasmids with correct sequencing were named Px459M-sgRNA5 and EZ-Guide-XH-sgRNA7, respectively.

[0042] The PCR primers used were as follows:

[0043] CAG-R: 5'-GTACTGGGCACAATGCCAG-3'

[0044] M13F: 5'-TGTAAAACGACGGCCAGT-3'

[0045] Plasmids Px459M-sgRNA5 and EZ-Guide-XH-sgRNA7 were double-digested with Hind III / Xho I, and the vector fragment Px459M-sTRIM56-KO-5 and the gene fragment sTRIM56-KO-7 were recovered from the gel. The vector and gene fragments were ligated overnight at 16°C and transformed into DH5α competent cells. The plasmids were then extracted and identified by Hind III / Xho I double digestion. The results are as follows: Figure 1 As shown in Figure C, the plasmid that tested positive was sent to Beijing Qingke Biotechnology Co., Ltd. for CAG-R sequencing. The recombinant plasmid that was correctly identified and sequenced was named Px459M-sTRIM56-KO, and the sequence inserted into the plasmid is shown in Sequence 1.

[0046] Example 2: Construction and identification of the porcine TRIM56 gene knockout cell line sTRIM56-KO-PAM-KNU

[0047] 2.1 Construction of the porcine TRIM56 gene knockout PAM-KNU cell line

[0048] PAM-KNU cells were cultured in RPMI 1640 cell culture medium containing a mixture of 5% FBS and 1% penicillin and streptomycin. PAM-KNU cells in good growth condition were seeded at 1 mL per well in 12-well plates and cultured overnight in a 5% CO2 incubator at 37°C. When cell confluence reached 80%–90%, PAM-KNU cell tolerance was tested at the following puromycin concentrations: 0, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 μg / mL. The lowest puromycin concentration that caused complete cell death on day 3 was considered the optimal drug concentration for PAM-KNU cell knockout cell line screening. The optimal puromycin concentration screened in this invention was 2.5 μg / mL.

[0049] PAM-KNU cells were seeded into 12-well plates, and when the cell density reached 80%–90%, they were... According to the P3000 instructions, the specific steps for transfecting Px459M-sTRIM56-KO plasmid into PAM-KNU cells are as follows: Remove Opti-MEM and culture medium containing 5% FBS from the 4°C freezer and place them in a 37°C water bath or preheat to room temperature; add 50 μL of Opti-MEM to a 1.5 mL centrifuge tube, then add 2 μL of P3000 and gently mix, then add 1 μg of Px459M-sTRIM56-KO recombinant plasmid, gently mix, and let stand at room temperature for 5 min; in another 1.5 mL centrifuge tube, add 50 μL of Opti-MEM, then add 3 μL of P3000... 3000 Transfection Reagent, mix gently; mix the liquid in two 1.5 mL centrifuge tubes, and place at room temperature for 15 min; replace the fresh culture medium in the 12-well plate, and add the liquid mixture into the 12-well plate, shake gently, and place in the cell incubator for continuous culture. At the same time, set the cells without transfection as a negative control.

[0050] After transfection for 24 h, continue to culture by adding culture medium containing 2.5 μg / mL puromycin, and after 3-4 days of screening, the control cells without transfection of plasmid all die, while the cells transfected with the knockout plasmid have live resistant clones. Replace the culture medium of the cells surviving after transfection of the knockout plasmid without puromycin for continuous culture. Dilute the cells by limiting dilution method, inoculate in a 96-well plate at 1-2 cells per well, observe the growth state of the single clone cells in each well, and according to the growth state, expand the cells in the 96-well plate to a 48-well plate, and after the 48-well plate is full, expand the cells in the 48-well plate to a 24-well plate, in the process, take part of the cells for identification, and continue to culture the remaining cells.

[0051] 2.2 RT-PCR identification

[0052] The cells taken out in the above process and wild-type PAM-KNU cells are extracted for total RNA by RNA-easy Isolation Reagent, and used as a template, and RT-PCR amplification is performed by using primers sTRIM56-F and sTRIM56-R for amplifying the sequence of pig TRIM56 gene, according to the instructions of HiScript II One Step RT-PCR Kit (Dye Plus), and the reaction system is as follows: One Step Enzyme Mix 2.5 μL, 2×One Step Mix 25 μL, sTRIM56-F 2 μL, sTRIM56-R 2 μL, RNA 1 μg, RNase Free ddH2O, and the total volume is 50 μL. After the system is prepared, it is mixed immediately, and placed in a PCR instrument for amplification reaction, and the reaction conditions are as follows: 50℃ for 30 min; 94℃ for 3 min; 94℃ for 30 s, 63℃ for 30 s, 72℃ for 2 min 20 s, 35 cycles; 72℃ for 7 min; 4℃ storage.

[0053] The RT-PCR primers used are as follows:

[0054] sTRIM56-F: 5'-ATGGTTTCCCAGGGCTCCTCA-3'

[0055] sTRIM56-R: 5'-TAACTGTCAGGAGGGCGGAC-3'

[0056] The positive clones were subjected to 1% agarose gel electrophoresis of RT-PCR amplification products, as shown in Figure 2 Lane 7: sTRIM56-KO-PAM-KNU cells. The electrophoretic band of the screened sTRIM56-KO-PAM-KNU cells was at 1076 bp, while that of the control PAM-KNU cells was at 2268 bp. The 1076 bp band of the sTRIM56-KO-PAM-KNU cells was recovered and sequenced, and the results are shown in Figure 2 Lane 7: sTRIM56-KO-PAM-KNU cells. The electrophoretic band of the screened sTRIM56-KO-PAM-KNU cells was at 1076 bp, while that of the control PAM-KNU cells was at 2268 bp. The 1076 bp band of the sTRIM56-KO-PAM-KNU cells was recovered and sequenced, and the results are shown in

[0057] 2.3 Western blotting identification

[0058] The sTRIM56-KO-PAM-KNU cells and wild-type PAM-KNU cells taken out in the above process were inoculated in 12-well plates, and the cells were collected after 48 h. The 12-well plates were placed on ice, and the supernatant was discarded. The cell samples were gently washed twice with pre-cooled PBS at 4°C, and cell lysis solution was added and supplemented with protease inhibitor PMSF (1:100). The cells were lysed on ice for 20 min, and the cells were shaken several times every 5 min to ensure complete lysis. The cells were scraped off with a gun head, and the samples were collected in 1.5 mL EP tubes. The samples were centrifuged at 4°C and 12000 r / min for 10 min, and the supernatant was mixed with an appropriate amount of loading buffer and boiled in a water bath for 15 min to denature the protein. The samples were subjected to 10% SDS-PAGE electrophoresis, and the proteins were transferred to NC membranes by wet transfer at 100 V for 70 min. The samples were blocked with 5% skim milk powder at room temperature for 2 h, and diluted TRIM56 antibody or internal reference β-actin antibody was used as the primary antibody, which was incubated at 4°C overnight. The samples were washed with TBST for 5 times, each for 5 min. HRP-labeled goat anti-rabbit IgG was used as the secondary antibody, which was incubated at room temperature for 1 h. The samples were washed with TBST for 5 times, each for 5 min. The developing solution was prepared according to the Sparkjade ECL super ultra-sensitive chemiluminescence kit instructions, and the protein was developed. The image was collected and analyzed by Bio-RAD gel imaging system.

[0059] Western blotting detection of sTRIM56 protein expression in PAM-KNU and sTRIM56-KO-PAM-KNU cells, as shown in Figure 2 Lane 7: sTRIM56-KO-PAM-KNU cells. The electrophoretic band of the screened sTRIM56-KO-PAM-KNU cells was at 1076 bp, while that of the control PAM-KNU cells was at 2268 bp. The 1076 bp band of the sTRIM56-KO-PAM-KNU cells was recovered and sequenced, and the results are shown in

[0060] Example 3: Application of pig TRIM56 knockout cell line in proliferation of PRRSV R98 vaccine strain

[0061] 3.1 Effect of sTRIM56 knockout on viral copy number

[0062] PAM-KNU cells and sTRIM56-KO-PAM-KNU cells were plated in 12-well plates, respectively, and when the cell density reached 90%, PRRSV R98 strain was diluted to MOI = 0.1 using RPMI 1640 cell culture medium containing 1% fetal bovine serum and 1% penicillin-streptomycin mixture, 1 mL per well in a 12-well plate, and incubated at 37°C, 5% CO2 in a cell incubator for 24h, 36h, 48h, and the cell supernatant was collected for PRRSV copy number and titer detection, and the cells were collected for PRRSV N protein level detection.

[0063] Total RNA was extracted from the cell supernatant sample using RNA-easy Isolation Reagent reagent, and reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR kit. The viral copy number was calculated according to the concentration of PRRSV-N plasmid, the plasmid concentration was adjusted and diluted by 10 times gradient dilution with ddH2O, and the dilution multiple was 10 10 ~ 10 3 copies / μL, using PRRSV-N-F / PRRSV-N-R primers, referring to the 2x RealStar Green Fast Mixture kit instructions, detecting PRRSV copy number by absolute fluorescent quantitative PCR, with reverse transcribed cDNA as template, and gradient diluted PRRSV-N plasmid positive standard, each group was repeated 3 times in parallel. The reaction system was: 2x RealStar Green Fast Mixture 10 μL, PRRSV-N-F 1 μL, PRRSV-N-R 1 μL, standard / cDNA 2 μL, ddH2O 6 μL. After the system was prepared, it was mixed and separated instantly, and was placed in a PCR instrument for amplification reaction, with reaction conditions of 95°C 1s; 95°C 5s, 55°C 20s, 72°C 20s, 40 cycles; 65°C 15s. The PRRSV copy number in each sample was obtained by comparing the Ct value of the standard curve, and the results were shown in Figure 3 A, the PRRSV R98 strain viral copy number in sTRIM56-KO-PAM-KNU cells was significantly higher than that in PAM-KNU cells at 24h, 36h, and 48h of viral infection (P<0.05).

[0064] The fluorescent quantitative PCR primers used were

[0065] PRRSV-NF: 5'-TGTGCCAAATGCTGGGTA-3'

[0066] PRRSV-NR:5'-GGGTAAAGTGATCCTGACG-3'

[0067] 3.2 Effect of sTRIM56 knockout on viral titer

[0068] MARC-145 cells were cultured in high-glucose DMEM medium containing a mixture of 10% FBS and 1% penicillin-streptomycin. MARC-145 cells were seeded into 96-well plates and cultured in a 5% CO2 incubator at 37°C. When cell confluence reached 90%, high-glucose DMEM medium containing a mixture of 1% FBS and 1% penicillin-streptomycin was used to culture the cell supernatants from cells infected with PRRSV R98 strain in section 3.1 at 24h, 48h, and 72h. -1 ~10 -10 Serial dilutions were performed, with three replicates for each dilution, using 100 μL per well. Cell supernatant was aspirated from the 96-well plates, and the diluted virus solution was added to the prepared MARC-145 cells in 96-well plates. A negative control group without added virus solution was included. Cytopathic effects were observed for 5 days, and TCID was calculated using the Reed-Muench method. 50 The result is as follows Figure 3 As shown in Figure B, at 24h, 36h, and 48h after viral infection, the viral titer of PRRSVR98 strain in sTRIM56-KO-PAM-KNU cells was significantly higher than that in PAM-KNU cells (P<0.05).

[0069] 3.3 Effect of sTRIM56 knockout on viral N protein

[0070] Cell samples infected with PRRSV R98 strain 24h, 36h, and 48h were collected as described in step 3.1. Following the Western blotting method in step 2.3, cell samples were collected and processed, and SDS-PAGE gel electrophoresis was performed. Diluted PRRSV N protein antibody and internal control β-actin antibody were used as primary antibodies, and HRP-labeled goat anti-mouse IgG and goat anti-rabbit IgG were used as secondary antibodies. Western blotting was performed to detect the PRRSV N protein level, and the protein bands were analyzed using ImageJ software. The results are shown below. Figure 3 As shown in C and 3D, at 24h, 36h and 48h after viral infection, the expression level of N protein of PRRSV in sTRIM56-KO-PAM-KNU cells was significantly higher than that in PAM-KNU cells (P<0.05).

[0071] 3.4 Data statistics

[0072] The data were analyzed using statistical methods of analysis of variance with Graphpad Prism 8.01 software, and the differences were determined by one-way repeated measures analysis of variance and least significant difference (LSD). P value < 0.05 was considered statistically significant.

[0073] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the examples, and any changes, modifications, combinations, substitutions, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A pig TRIM56 gene knockout cell line as an application in a pig PRRSV virus or pig PRRSV virus vaccine production cell line, characterized in that The pig TRIM56 gene knockout cell line promotes proliferation of the pig PRRSV R98 strain, increases viral titer, or promotes expression of N protein of the pig PRRSV R98 strain, and the cell line is a PAM-KNU cell line, and the nucleotide sequence of the pig TRIM56 is shown in GenBank Accession No. KJ881160.

2. Use according to claim 1, characterized in that The pig TRIM56 gene coding region from 929 to 2120 of the pig TRIM56 gene knockout cell line is deleted by 1192 bases.

3. Use according to claim 1, characterized in that The pig TRIM56 gene knockout cell line is obtained by the following steps: After the pig TRIM56 gene knockout plasmid Px459M-sTRIM56-KO is transfected into cells, the pig TRIM56 gene knockout cell line sTRIM56-KO-PAM-KNU is obtained after screening, culture, and identification. The pig TRIM56 gene knockout plasmid Px459M-sTRIM56-KO is a nucleotide molecule in which a nucleotide sequence as shown in SEQ ID NO: 1 is inserted between the Hin d III enzyme cleavage sites and the farthest Hin d III enzyme cleavage sites Bbs I enzyme cleavage sites.

4. Use according to claim 3, characterized in that The pig TRIM56 gene knockout plasmid Px459M-sTRIM56-KO is prepared by the following steps: (1) Design two pairs of guide RNA primers for the pig TRIM56 gene, and the sequences are as follows: TRIM56-KO-5F: 5'-GGCGGTCGCCTTTGCCCGTAGTTT-3' TRIM56-KO-5R: 5'-AAACTACGGGCAAAGGCGACCGCC-3' TRIM56-KO-7F: 5'-GTCGTGATCCTCGATCCCAAGTTT-3' TRIM56-KO-7R: 5'-AAACTTGGGATCGAGGATCACGAC-3' The two pairs of guide RNA primers are phosphorylated and annealed to form sticky end double-stranded TRIM56-KO-5 and TRIM56-KO-7. (2) Construct the knockout plasmid Px459M-sTRIM56-KO The Px459M vector and the EZ-Guide-XH vector were respectively digested with Bbs After enzyme digestion, the two pairs of guide RNA primers were respectively connected, the plasmids were transformed and extracted, and the recombinant plasmids Px459M-sgRNA5 and EZ-Guide-XH-sgRNA7 were respectively obtained; The recombinant plasmids Px459M-sgRNA5 and EZ-Guide-XH-sgRNA7 were respectively digested with Hin d III / Xho I were digested with double enzymes, and the vector fragment Px459M-sTRIM56-KO-5 and the gene fragment sTRIM56-KO-7 were recovered from the gel. The vector fragment Px459M-sTRIM56-KO-5 and the gene fragment sTRIM56-KO-7 were connected and transformed to obtain the pig TRIM56 gene knockout plasmid Px459M-sTRIM56-KO.

5. Use according to claim 4, characterized in that The phosphorylation and annealing conditions are as follows: The reaction system is: TRIM56-KO-5F / TRIM56-KO-7F 1 μL, TRIM56-KO-5R / TRIM56-KO-7R 1 μL, 10 × T4 PNK Buffer 1 μL, T4 PNK 1 μL, ddH2O 6 μL; the reaction conditions are: 37 ℃ for 30 min, 95 ℃ for 5 min, cooling to 25 ℃ at a gradient of-0.1 ℃ / s, 25 ℃ for 5 min, and 4 ℃ for 5 min.

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