CN110862968A - Construction method of PK-15 cell line with knockout of MAP3K8 gene and its application - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a PK-15 cell line knocked out by a MAP3K8 gene. The preservation number of the PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knockout is CCTCC NO: C2019176. The construction method of the cell line comprises the following steps of 1: respectively designing two sgRNAs according to a swine MAP3K8 gene sequence, and connecting the double-stranded sg RNA with a digested lentiGuide-EGFP vector to obtain a recombinant lentivirus expression plasmid lentiGuide-EGFP-MAP3K8-sg RNA; 2: co-transfecting the recombinant lentivirus expression plasmid and the virus packaging helper plasmid to a PK-15 cell; 3: collecting virus liquid, filtering, ultracentrifuging, concentrating and purifying virus; 4: infecting PK-15 cells with lentiviruses with multiplicity of infection MOI =30, and then performing single cell sorting with an ultra-speed flow cytometric sorting system; 5: and carrying out amplification culture and sequencing identification on the sorted monoclonal cells. The cell line can promote the proliferation of FMDV and SVV, improve the virus yield, can be used for large-scale cell culture and production of FMDV and SVV vaccine strains, and can provide a powerful tool for researching the action mechanism of MAP3K8 in the virus infection process.

Description

Construction method of PK-15 cell line with knockout of MAP3K8 gene and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a PK-15 cell line with a knockout of the MAP3K8 gene and its application.

Background technique

Pig kidney epithelial cell PK-15, derived from pig kidney, Chinese name is pig kidney epithelial cell. The cells are sensitive to a variety of viruses, such as porcine circovirus (PCV), porcine parvovirus (PPV), swine fever virus (CSFV), etc., and can be applied to porcine circovirus vaccine, porcine parvovirus vaccine, swine fever virus Preparation of vaccines, etc.

TPL2, a serine/threonine kinase, also known as COT or MAP3K8, forms a complex with p105 and ABIN2 to remain inactive when unstimulated (Gantke, T., S. Sriskantharajah, and S.C. . Ley, Regulation and function of TPL-2, an IkappaB kinase-regulated MAPkinase kinasekinase. Cell Res, 2011. 21(1): p. 131-45.). When stimulated, it can activate downstream ERK, JNK, p38 and NF-κB and other signal transduction pathways through various receptors such as TLR, TNFR and IL1R; it can also stimulate macrophages, dendritic cells, neutrophils Granulocytes and other innate immune cells produce a large number of cytokines such as IFN-γ and TNF-α (Gantke T, S.S., Sadowski M, Ley SC., IκB kinase regulation of the TPL-2/ERK MAPKpathway. 2012.). TPL2 is also essential in regulating the differentiation of CD4+ T cells into distinct Th cell lineages, and is an important player in innate immunity, inflammation, and tumors.

CRISPR-Cas9 gene editing technology is the third-generation genome editing technology developed rapidly after ZFN and TALEN technology. The technology is derived from the CRISPR-Cas acquired immune system that resists phage invasion in bacteria and archaea, and has been gradually developed through artificial transformation (Li Peizhe, Sichuan Agricultural University. Development and Application of CRISPR/Cas9 Technology [N]. Scientific Reports , 2019-08-20 (B02).). With the help of CRISPR and Cas9, bacteria can target and silence key parts of the invader's genetic information via the guidance of small RNA molecules. The CRISPR-Cas9 genome editing technology specifically recognizes the target gene sequence through a segment of gRNA, and guides the Cas9 endonuclease to cut double-stranded DNA at the target site, followed by reconnection by the cell's non-homologous end joining repair mechanism (NHEJ). Breaks in genomic DNA and introduces insertion or deletion mutations. Compared with ZFN and TALEN technology, the gene editing efficiency of CRISPR-Cas9 system is higher, and the vector construction and use of Cas9 system is also more convenient, and it has been applied in various species. It is currently the most widely used gene editing technology.

SUMMARY OF THE INVENTION

The invention utilizes CRISPR-Cas9 gene editing technology to construct a PK-15 cell line PK-15-KO-MAP3K8 knocking out the MAP3K8 gene, and the cell line can promote the proliferation of foot-and-mouth disease virus (FMDV) and Seneca Valley virus (SVV), Increase virus yield. It can be used for large-scale cell culture and production of FMDV and SVV vaccine strains, and can provide a powerful tool for studying the mechanism of action of MAP3K8 during virus infection.

In the first aspect, the present invention provides a PK-15 cell line PK-15-KO-MAP3K8 with a knockout of the MAP3K8 gene, whose deposit number is CCTCC NO: C2019176.

In the second aspect, the present invention also provides a method for constructing a PK-15 cell line PK-15-KO-MAP3K8 knocking out the MAP3K8 gene, which specifically includes the following steps:

Step 1: Primer design and plasmid construction: According to the porcine MAP3K8 gene sequence, two sgRNAs were designed respectively, and the double-stranded sgRNA was ligated with the digested lentiGuide-EGFP vector to obtain the recombinant lentivirus expression plasmid lentiGuide-EGFP-MAP3K8 -sgRNA;

Specifically, the double-stranded sg RNA was ligated with the digested lentiGuide-EGFP vector using T4 DNA ligase.

Step 2: Plasmid transfection: co-transfect lentiGuide-EGFP-MAP3K8-sg RNA lentivirus expression plasmid and virus packaging auxiliary plasmid into PK-15 cells;

Specifically, the transfection process was performed according to the instructions of Lipofectamine 2000 transfection reagent.

Before transfection, PK-15 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 0.2 mg/mL streptomycin and 200 IU/mL penicillin.

Step 3: Lentivirus concentration: collect the virus solution, filter, ultracentrifuge, concentrate and purify the virus, and save it for future use.

Lentivirus titer determination before lentivirus infection: PK-15 cells were infected with the virus stock solution after serial dilution, and the virus titer was calculated.

Step 4: Lentivirus infection and flow sorting: PK-15 cells were infected with lentivirus with a multiplicity of infection MOI=30, and then single-cell sorting was carried out with an ultra-fast flow cytometry sorting system.

Step 5: Culture of monoclonal cells and identification by sequencing: the sorted monoclonal cells are expanded and cultured and sequenced to identify single cell clones.

Specifically, the expanded culture is as follows: inoculating the sorted monoclonal cells into a 96-well plate, subculture to a 48-well plate after full growth, and sequentially expanding the culture to a 24-well plate, a 12-well plate, a 6-well plate, and a 24-well plate. in a T25 flask.

Further, the sequence of the sgRNA is:

P-MAP3K8-sgRNA1 Forward: 5'-CACCGTTCCATAATGTCTATTACAT-3',

P-MAP3K8-sgRNA1Reverse: 5'-AAACATGTAATAGACATTATGGAAC-3';

P-MAP3K8-sgRNA2Forward: 5'-CACCGAGATCCCAGATTCCTGGGGT-3',

P-MAP3K8-sgRNA2Reverse: 5'-AAACACCCCAGGAATCTGGGATCTC-3';

The lentiGuide-EGFP vector described in step 1 was digested with BsmBI enzyme and then ligated with sg RNA.

In the third aspect, the present invention provides the application of the PK-15 cell line PK-15-KO-MAP3K8 knocking out the MAP3K8 gene in the study of the mechanism of virus infection.

Further, the virus is FMDV or SVV.

In a fourth aspect, the present invention provides the application of the PK-15 cell line PK-15-KO-MAP3K8 knocking out the MAP3K8 gene in the isolation and culture of FMDV or SVV.

In the fifth aspect, the present invention provides the application of the PK-15 cell line PK-15-KO-MAP3K8 knocking out the MAP3K8 gene in the large-scale cellular culture and production of FMDV or SVV vaccine strains.

The present invention has the following beneficial effects:

1. The present invention uses the CRISPR-Cas9 system to construct a PK-15 cell line PK-15-KO-MAP3K8 that knocks out the MAP3K8 gene. The establishment of this cell line provides a powerful tool for studying the mechanism of action of MAP3K8 in the process of virus infection.

2. The PK-15 cell line PK-15-KO-MAP3K8 with knockout of MAP3K8 gene cannot express MAP3K8 protein correctly due to frameshift mutation caused by insertion or deletion of certain fragments. Due to the antiviral effect of MAP3K8 protein, FMDV virus and SVV virus cannot proliferate in wild-type PK-15 cells. However, the PK-15 cell line PK-15-KO-MAP3K8 knocking out the MAP3K8 gene cannot express the MAP3K8 protein correctly, so it is conducive to the proliferation of FMDV virus or SVV virus in this cell line, and obtains a larger amount of high titer virus. It can be used for large-scale cell culture and production of FMDV or SVV vaccine strains.

Description of drawings

Figure 1 is a structural diagram of the lentiGuide-EGFP-MAP3K8-sg RNA lentiviral expression vector constructed in the present invention. a is lentiGuide-EGFP-MAP3K8-sg RNA1, b is lentiGuide-EGFP-MAP3K8-sg RNA2.

Figure 2 is a diagram of the transfection of lentiGuide-EGFP-MAP3K8-sg RNA lentiviral expression vector into PK-15 cells under a fluorescence microscope in the present invention (transmitted light and fluorescence detection, ×200).

Fig. 3 is the analysis diagram of the insertion and deletion mutants of the MAP3K8 gene of clones #1, #19 and #20 of the PK-15-KO-MAP3K8 cell line of the present invention. a is clone #1, b is clone #19, c is clone #20.

Figure 4 is a graph showing the protein abundance of MAP3K8 in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells detected by Western blotting in the present invention.

Figure 5 is a graph showing the absolute quantitative detection of FMDV virus copy number in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells according to the present invention.

Figure 6 is a graph showing the absolute quantitative detection of SVV virus copy number in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells according to the present invention.

Figure 7 is a graph showing the relative quantitative detection of the transcription level of FMDV replication in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells in the present invention.

Figure 8 is a graph showing the relative quantitative detection of the transcription level of SVV replication in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells in the present invention.

Figure 9 is the protein abundance map of the Western blotting detection of FMDV replication in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells in the present invention.

Figure 10 is the protein abundance map of Western blotting detection of SVV replication in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells in the present invention.

Figure 11 is a graph showing the fluorescence expression of FMDV and the corresponding optical density values in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells observed under a fluorescence microscope in the present invention.

Figure 12 is a graph showing the fluorescence expression of SVV and the corresponding optical density values in PK-15-WT-MAP3K8 and PK-15-KO-MAP3K8 cells observed under a fluorescence microscope in the present invention.

In the figure, WT represents the control PK-15-WT-MAP3K8 cell line, and KO represents the PK-15-KO-MAP3K8 cell line.

* in the figure represents p<0.05; *** represents p<0.001.

Deposit information:

Preservation time: July 23, 2019;

Name of the depositary unit: China Type Culture Collection;

Deposit number: CCTCC NO: C2019176;

Depositary address: Wuhan University, Wuhan, China;

Classification name: porcine kidney epithelial cell PK-15-KO-MAP3K8.

Detailed ways

The present invention will be further elaborated below with reference to the accompanying drawings and specific embodiments of the description, and the embodiments are only used to explain the present invention, but not to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are all commercially available reagents and materials unless otherwise specified.

Sources of materials used in the examples:

Cells and viruses: Pig kidney cells (PK-15) were purchased from Kunming Institute of Zoology, Chinese Academy of Sciences. Pig kidney cells (PK-15) were cultured in medium containing 10% DMEM supplemented with 10% heat-inactivated fetal bovine serum (HyClone), 1% streptomycin (0.2 mg/mL) and penicillin (200 IU/mL) ). All cells were maintained at 37 °C in a constant temperature incubator with ₅ % CO. Foot-and-mouth disease strain A/GDMM/CHA/2013 and swine Seneca Valley virus (SVV) strain were preserved by the foot-and-mouth disease epidemiology team of Lanzhou Veterinary Research Institute.

Reagents and antibodies: 0.25% EDTA-Trypsin, Opti-MEM, DMEM medium and fetal bovine serum were purchased from Gibco; Lipofectamine 2000, lipofection reagent, SDS-PAGE protein loading buffer (5×), 0.1 MDTT and reverse transcriptase M-MLV were purchased from Life Invitrogen Company; cell culture consumables such as cell culture flasks, culture plates and pipettes were purchased from Coring Company; Phosphate buffered saline (PBS solution pH7.4, 0.0067M) Purchased from Hyclone Company; RAPI cell lysate and PMSF were purchased from Biyuntian Company; Protein Marker was purchased from Thermo Scientific Company; RNA extraction reagents Trizol, SYBR Permix Ex Taq II, Trans5α competent, LA Taq DNA polymerase, restriction Endonuclease BsmBI, T4 DNA ligase, RNase inhibitor (RRI), Oligo (dT) primers, Random primers, deoxyribonucleoside triphosphates (dNTPs) and nucleic acid markers were purchased from Bao Bioengineering Dalian Co., Ltd. ; The qRT-PCR primers used to detect CRISPR/Cas9 gene knockout efficiency were synthesized by Aoke Biotechnology Co., Ltd. Commercial antibodies used in the present invention include: HRP-labeled goat anti-rabbit IgG antibody (Proteintech), HRP-labeled goat anti-mouse IgG antibody (Proteintech), anti-MAP3K8 polyclonal antibody (Santa Cruz Biotechnology), anti-β-actin Monoclonal antibody (Santa Cruz Biotechnology). Anti-FMDV polyclonal antibody and anti-SVV polyclonal antibody were prepared by the foot-and-mouth disease epidemiology team of Lanzhou Veterinary Research Institute. The preparation method is referred to (Chen Fei. Preparation and identification of polyclonal antibody against foot-and-mouth disease virus 3C protein [D]. Shandong Agricultural University, 2018 .) and (Hu Tao et al. Analysis of the results of preparing rabbit polyclonal antibodies with purified vesicular stomatitis virus antigen for teaching [J], Health Vocational Education, 2019(6): 112-114). Immunofluorescence rabbit secondary antibody was purchased from CellSignaling Technology (CST).

Instruments: ABI gradient PCR instrument, CO ₂ constant temperature incubator, QuanStudio3/5 quantitative PCR instrument, and 700 series -80°C refrigerator were purchased from Thermo Company in the United States; laser confocal microscope was purchased from LEICA Company in Germany; GLOMAX chemiluminescence detector was purchased from PROMEGA company; electric heating constant temperature water bath was purchased from Shanghai Yiheng Instrument Co., Ltd.; 5804R high-speed refrigerated centrifuge and small high-speed centrifuge were purchased from Germany Eppendorf company; gel imager was purchased from American Bio-Rad company; ice maker was purchased from FOCUSUN Company; UV-Vis spectrophotometer was purchased from Shanghai Angla Instrument Company; DYY-12 stabilized electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory; biological safety cabinet was purchased from Suzhou Sujing Group.

The present invention will be described in detail below with reference to the embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those skilled in the art, several adjustments and improvements can be made without departing from the inventive concept. These all belong to the protection scope of the present invention.

1. Primer design and plasmid construction

Referring to the porcine MAP3K8 gene (Gene ID 100622217) in the NCBI database, the protein sequence was analyzed and the conserved region in Exon2 was selected for target screening, and the Ensembl genome database (http://asia.ensembl.org/Sus_scrofa/Transcript /Summary?db=core; g=ENSSSCG00000020705; r=10:40709927-40746355; t=ENSSSCT00000033089), according to the Cas9 target design principle, the higher score was selected as the sg RNA candidate sequence, and two Exon2 targets were designed respectively. After adding the complementary sticky ends generated by BsmBI site cleavage at both ends of the sgRNA, the upstream and downstream sgRNA sequences were designed and obtained, as shown in Table 1. The double-stranded sg RNA was mixed with the digested lentiGuide-EGFP vector (lentiviral expression vector lentiGuide-EGFP vector was provided by Southern Model Biotechnology Co., Ltd.), ligated at 16 °C overnight under the action of T4 DNA ligase, and the MAP3K8 sgRNA Oligo double-stranded DNA was ligated into a linearized vector. The ligation reaction solution was transformed into Trans5α competent cells, spread on an ampicillin-resistant plate, overnight at 37°C. Pick the monoclonal bacteria, culture them in LB medium with ampicillin resistance, shake culture in a 37°C incubator, and extract plasmids after 12 hours. After the construction of the vector was completed, it was sent to Xi'an Qingke Jersey Biotechnology Co., Ltd. for sequencing and identification. The structures of the constructed lentiGuide-EGFP-MAP3K8-sg RNA lentiviral expression plasmids lentiGuide-EGFP-MAP3K8-sg RNA1 and lentiGuide-EGFP-MAP3K8-sg RNA2 are shown in Figure 1.

Table 1 Primer sequences of porcine MAP3K8-sg RNA

primer name Primer sequence (5'-3') P-MAP3K8-sgRNA1 Forward CACCGTTCCATAATGTCTATTACAT P-MAP3K8-sgRNA1 Reverse AAACATGTAATAGACATTATGGAAC P-MAP3K8-sgRNA2 Forward CACCGAGATCCCAGATTCCTGGGGT P-MAP3K8-sgRNA2 Reverse AAACACCCCAGGAATCTGGGATCTC

2. Cell Culture and Plasmid Transfection

PK-15 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 0.2 mg/mL streptomycin and 200 IU/mL penicillin. Incubate at 37°C in a saturated humidity incubator with constant temperature and 5.0% CO ₂ gas. The PK-15 cells in good growth state were inoculated into a 6-well cell culture plate, and 1×10 ⁵ cells were passed into each well. When the cell density reached 70% to 80%, transfection was performed according to Lipofectamine 2000. Reagent Instructions The lentiGuide-EGFP-MAP3K8-sg RNA lentiviral expression plasmids (lentiGuide-EGFP-MAP3K8-sg RNA1 and lentiGuide-EGFP-MAP3K8-sg RNA2) and virus packaging helper plasmids were co-transfected into PK-15 cells. Plasmid and Lipofectamine 2000 reagent (both according to the ratio of 1 μg: 2 μl) were added to Opti-MEM respectively. After the two were mixed and allowed to stand for 15 min, the Opti-MEM mixture was directly added to the cells; the cells were placed at 37°C again. , in a 5% CO ₂ incubator. After 48 hours, the fluorescence expression was observed under a microscope, and it was found that a large amount of fluorescent protein was expressed, indicating that the plasmid was successfully transfected into PK-15 cells, as shown in Figure 2.

3. Virus concentration

The virus (ie, unpurified cell supernatant) was harvested for the first time 24 hours after transfection, placed in a 4°C refrigerator, and the medium was replaced in the dish to continue culturing. The virus was harvested a second time 48h after transfection and combined with the supernatant from the first collection. The collected virus solution was filtered using a 0.22 μm filter membrane, concentrated and purified by ultracentrifugation at 4°C, and stored in aliquots.

4. Lentiviral titer determination

The day before the assay, PK-15 cells were seeded into 96-well plates, and the cell status was confirmed under a microscope before virus infection. The cell density was uniform, the shape was normal, and there was no contamination. After the virus stock solution was serially diluted, it was added to 96-well plates in turn, and then placed in a CO ₂ incubator after labeling. About 24 hours after virus infection, in accordance with the principle of low concentration group to high concentration group, fluids were added in turn, and the culture was continued. 72h after infection, the medium containing puromycin was replaced, and after culturing for 24h, all groups were photographed and the virus titer was calculated. Virus titer (TU/mL) = number of GFP positive cells/dilution factor. The titer of the virus constructed in this example was 1×10 ⁹ TU/mL.

5. Lentivirus infection and flow sorting

The PK-15 cells in good growth state were seeded in 6-well plates, 1×10 ⁵ cells per well, and when the cell density reached 70% to 80%, the infection was carried out according to the multiplicity of infection of lentivirus (MOI=30). The cell supernatant was discarded, a mixture of lentiGuide-EGFP-MAP3K8-sg RNA lentivirus and serum-free medium was added, and Polybrene was added to a final concentration of 6 μg/mL. After 12 h, the cell supernatant was replaced with fresh DMEM medium, and the culture was continued for 72 h. After washing the cells twice with PBS, 0.25% EDTA-Trypsin was added, digested at 37° C. for 2 min, 800 rpm, 5 min, and 1 mL of DMEM medium was added to prepare a single cell suspension. Single-cell sorting was performed using an ultra-fast flow cytometry system.

6. Culture and sequencing of monoclonal cells

The sorted monoclonal cells were inoculated into a 96-well plate, and the medium was changed every 2 to 3 days. When the 96-well plate was full, it was passaged to a 48-well plate, and then expanded to 24-well plate, 12-well plate, and 6-well plate. plates and T25 flasks. The expanded monoclonal cells were transferred to a 6-well plate at a density of 1×10 ⁵ , and the cells were harvested when the cell density reached about 90%. Total RNA of monoclonal cell lines was extracted by Triozl lysis method, and reverse transcribed into cDNA template for PCR amplification. Then, primers were designed to amplify the full length of the MAP3K8 gene, and the primer sequences are shown in Table 2. The PCR product was sent to Xi'an Qingke Jersey Biotechnology Co., Ltd. for sequencing and identification of base insertion or deletion. A total of 23 positive clones were selected for sequence analysis. After sequencing, sequence alignment was performed with the MAP3K8 gene sequence in wild-type cell lines to check for insertion or deletion mutations. The sequencing results showed that the DNA sequences of the positive clones had certain insertions and deletions, indicating that the lentiviruses carrying Cas9 and MAP3K8 sg RNA1+sg RNA2 constructed in this example had effectively cut the MAP3K8 gene in PK-15 cells at specific sites. This leads to mismatch repair in the genome of the cell. Among the 23 selected monoclonal clones, the positive clones #1, #19 and #20 were monoclonal cell lines. MAP3K8 knockout monoclonal cell line #1 has a 2-base insertion and a 2-base deletion; MAP3K8 knockout monoclonal cell line #19 has a 3-base deletion; MAP3K8 knockout monoclonal Cell line #20 has a 2 base deletion, see Figure 3.

Table 2 PCR amplification primers of porcine MAP3K8 gene

primer name Primer sequence (5'-3') MAP3K8 Forward GGACAGCAGGTGAAACGCATCT MAP3K8Reverse CCACAGCCATAGCCACAACC

7. Validation of PK-15-KO-MAP3K8 protein level in PK-15 cell line knocking out MAP3K8 gene

In order to verify whether the knockdown of MAP3K8 has an effect on the protein expression level of the PK-15-KO-MAP3K8 cell line, Western blotting was used to detect the protein expression in the PK-15-KO-MAP3K8 cell line and the control PK-15-WT-MAP3K8 cell line. MAP3K8 protein expression levels. After the cells were expanded and cultured, the cells were washed with PBS, and RAPI cell lysate containing PMSF and 1× protein loading buffer were added. The cells were scraped and placed in a metal bath at 98 °C for 10 min, and then centrifuged at 10,000 rpm for 10 min, and the supernatant was taken. After separation by 10% SDS-PAGE and transfer electrophoresis, the protein bands separated by electrophoresis were transferred to NC membrane. The NC membrane was blocked with 5% nonfat milk powder solution at room temperature for 1-1.5 h; MAP3K8 antibody (1:1000 dilution) and β-actin antibody (1:5000 dilution) were incubated with NC membrane at room temperature overnight; after washing and incubation with 1×TBST NC membrane 4 times (8min/time); then incubated with HRP-labeled goat anti-rabbit IgG (1:5000 dilution) and goat anti-mouse IgG (1:5000 dilution) secondary antibody incubation solution and NC membrane at room temperature for 1h; The NC membrane was washed 4 times with 1 × TBST (8 min/time); the electrophoresis results were photographed and analyzed using an automatic chemiluminescence imaging analysis system. The results showed that the expression of MAP3K8 in wild-type PK-15-WT-MAP3K8 cells was normal, while the expression of MAP3K8 was not detected in the MAP3K8 knockout monoclonal cell lines #1, #19 and #20, and the internal reference β-actin expression was normal. And basically the same, as shown in FIG. 4 , indicating that the MAP3K8 gene knockout cell line was successfully constructed in this example.

8. Cell culture and inoculation

PK-15-KO-MAP3K8 and PK-15-WT-MAP3K8 cells were cultured in DMEM medium containing 10% fetal bovine serum, passaged for 2 to 3 generations after self-recovery, digested and seeded in 6 wells after the cells were stable. In the plate, 1×10 ⁵ cells per well were placed in a 37°C, 5% CO ₂ incubator. When the cells grew to 70% to 90%, the cells were washed twice with serum-free DMEM to remove the cells. residual serum. Dilute the venom with serum-free DMEM, then add the diluted venom to the cells according to an appropriate volume, incubate at 37°C in a 5% CO ₂ incubator for 1 h, discard the venom and replace it with 2% DMEM maintenance solution, Continue to cultivate.

9. Absolute quantitative verification of the replication of FMDV and SVV on PK-15-KO-MAP3K8

Cells were harvested at 0h, 8h, and 16h after FMDV and SVV inoculation, and the total RNA of cells was extracted by Triozl lysis method, and then absolute quantitative primers FMDV-3D-F/R, SVV-3D- F/R, and probes for 3D proteins of FMDV and SVV were subjected to absolute quantitative detection of the extracted whole genome RNA, and the copy number of FMDV and SVV was determined. The primer sequences are shown in Table 3. It can be seen from Figure 5 that in the samples collected at 8h and 16h after inoculation, the copy number of FMDV in PK-15-KO-MAP3K8 cells is much higher than that in PK-15-WT-MAP3K8 cells; Figure 6 shows that the copy number of SVV in PK-15-KO-MAP3K8 cells is much higher than that in PK-15-WT-MAP3K8 cells. This is because MAP3K8 protein has an antiviral effect, and PK-15-KO-MAP3K8 cannot express MAP3K8 protein correctly, so it is beneficial to the proliferation of FMDV and SVV viruses. That is to say, compared with PK-15-WT-MAP3K8, PK-15-KO-MAP3K8 cells replicated more FMDV and SVV after FMDV and SVV infection. This indicates that PK-15-KO-MAP3K8 cells promote the replication of FMDV and SVV viruses by knocking down MAP3K8.

Table 3 Absolute quantitative primer sequences

primer name Primer sequence (5'-3') FMDV-3DForward CACTGGTGACAGGCTAAGG FMDV-3D Reverse CCCTTCTCAGATTCCGAGT FMDV 3D Probe FAM-TCCTTTGCACGCCGTGGGAC-TAMRA SVV-3D Forward AGAATTTGGAAGCCATGCTCT SVV-3DReverse GAGCCAACATAGARACAGATTGC SVV3D probe FAM-TTCAAACCAGGAACACTACTCGAGA-BHQ1

10. Relative quantitative verification of the replication of FMDV and SVV on PK-15-KO-MAP3K8

Cells were harvested at 0h, 8h, and 16h after FMDV and SVV inoculation, and the total RNA of cells was extracted by Triozl lysis method, and reverse transcribed into cDNA for relative quantitative verification. The relative quantitative primers FMDV-F/R and SVV-F/R for amplifying FMDV and SVV3D proteins were used to perform relative quantitative detection on the reverse transcribed cDNA, respectively, to determine the changes in the transcription levels of FMDV and SVV. The primer sequences are shown in Table 4. The results are shown in the figure, in the samples collected at 8h and 16h after exposure, the FMDV mRNA expression in PK-15-KO-MAP3K8 cells was much higher than that in PK-15-WT-MAP3K8 cells (Fig. 7); SVV mRNA expression in PK-15-KO-MAP3K8 cells was much higher than that in PK-15-WT-MAP3K8 cells (Fig. 8). That is to say, PK-15-KO-MAP3K8 cells can significantly promote the replication of FMDV and SVV compared with PK-15-WT-MAP3K8 cells, which is consistent with the absolute quantitative results.

Table 4 qPCR primer sequences

primer name Primer sequence (5'-3') P-GAPDHForward ACATGGCCTCCAAGGAGTAAGA P-GAPDHReverse GATCGAGTTGGGGCTGTGACT FMDVForward TGGGACCATACAGGAGAAGT FMDVReverse GTAGCTTGGAATCTCGAAGAGG SVVForward AGAATTTGGAAGCCATGCTCT SVVReverse GAGCCAACATAGARACAGATTGC

11. Western blotting to verify the replication of FMDV and SVV on PK-15-KO-MAP3K8

Cells were harvested at 0h, 8h, and 16h after inoculation with FMDV and SVV. The PK-15-KO-MAP3K8 and PK-15-WT-MAP3K8 cell samples were treated with the above method, and Western blotting experiments were performed. The results are shown in the figure, in the samples collected at 8h and 16h after inoculation, the abundance of FMDV structural proteins VP1, VP2 and VP3 in PK-15-KO-MAP3K8 cells was much higher than that of PK-15-WT-MAP3K8 The abundance of structural proteins VP1, VP2 and VP3 of FMDV in cells (Fig. 9); the abundance of structural proteins VP1, VP2 and VP3 of SVV in PK-15-KO-MAP3K8 cells was much higher than that of PK-15-WT-MAP3K8 Abundance of SVV structural proteins VP1, VP2 and VP3 in cells (Figure 10).

12. Indirect immunofluorescence experiment

PK-15-KO-MAP3K8 and PK-15-WT-MAP3K8 cells were plated in 20mm glass dishes, and when the cells grew to 70% to 80%, they were infected with FMDV and SVV viruses at MOI=2; After 0h, 8h, and 16h, the cells were harvested and washed 3 times with 1×PBS; fixed with 4% paraformaldehyde (1 mL per dish) for 1 h at room temperature in the dark; washed with 1×PBS for 3 times, 5min/time (add 1 ×PBS to prevent cells from being washed up); permeabilize with 0.2% Triton X-100 (100mL PBS+200μL Triton100) for 1h at room temperature; wash 3 times with 1×PBS, 5min/time; use 5%BSA (5%BSA: 10mL) +0.5g BSA) block at 37℃ for 1h; aspirate the blocking solution, add primary antibody diluted with 5% BSA, overnight at 4℃; wash three times with 1×PBST, 10min/time, add 1×PBST (1×PBST: 100mL) Fluorescein-labeled secondary antibody (immunofluorescence rabbit secondary antibody) diluted in 1×PBS+50μLTWeen20), incubated at 37°C for 1h; washed three times with 1×PBST, 10min/time, and 100μL of anti-fluorescence decay mounting medium was added to each dish to mount (containing DAPI); use a laser confocal instrument to observe the fluorescence, and save the picture. The results are shown in the figure, in the samples collected at 8h and 16h after inoculation, the fluorescence expression levels of FMDV VP1, VP2 and VP3 virus particles in PK-15-KO-MAP3K8 cells were much higher than those of PK-15-WT. -Fluorescence expression of FMDVVP1, VP2 and VP3 virus particles in MAP3K8 cells (Fig. 11); Fluorescence expression of SVVVP1, VP2 and VP3 virus particles in PK-15-KO-MAP3K8 cells was much higher than that of PK-15-WT- Fluorescence expression of VP1, VP2 and VP3 virions of SVV in MAP3K8 cells (Fig. 12). The fluorescence expression levels of FMDV and SVV were then quantitatively analyzed by ImagePro-Plus software.

13. Determination of virus titer TCID ₅₀

FMDV and SVV were infected with PK-15-KO-MAP3K8 and PK-15-WT-MAP3K8 cells respectively, and the virus venom was collected when the cytopathic variable reached 50%-60%. After repeated freezing and thawing three times, the obtained FMDV and SVV viruses were serially diluted ^10-2 to ^10-9 times with serum-free DMEM, and the PK- 15-KO-MAP3K8 and PK-15-WT-MAP3K8 cells were seeded in 8 wells at each dilution with 0.1 mL per well. Placed in a 37°C, 5% CO ₂ constant temperature incubator and observed for 4 days, observed and recorded cytopathic changes (CPE) every half day, and calculated the TCID ₅₀ of the amplified virus according to the Reed-Muench method. The TCID ₅₀ of FMDV virus was determined by the Reed-Muench method, the TCID ₅₀ of PK-15-WT-MAP3K8 cells was 10 ^-3.6 /0.1mL, and the TCID ₅₀ of PK-15-KO-MAP3K8 cells was 10 ^-4.58 /0.1 mL, see Tables 5-6. The TCID ₅₀ of the SVV virus was determined by the Reed-Muench method, and the TCID ₅₀ of the PK-15-WT-MAP3K8 cells was 10 ^-6.8 /0.1 mL. The TCID ₅₀ determination result of PK-15-KO-MAP3K8 cells was 10 ^-8.4 /0.1 mL, as shown in Tables 7-8. It can be seen that compared with PK-15-WT-MAP3K8 cells, higher titers of FMDV and SVV can be obtained using PK-15-KO-MAP3K8 cells.

Table 5 TCID ₅₀ of FMDV expanded from PK-15-WT-MAP3K8 cells

Table 6 TCID ₅₀ of FMDV expanded in PK-15-KO-MAP3K8 cells

Table 7 TCID ₅₀ of SVV expanded from PK-15-WT-MAP3K8 cells

Table 8 TCID ₅₀ of SVV expanded in PK-15-KO-MAP3K8 cells

Specific embodiments of the present invention have been described above. It is to be understood that the present invention is not limited to the specific embodiments described above. The above-mentioned embodiments are only preferred embodiments of the present invention, and are only used to explain the present invention, but not to limit the scope of implementation of the present invention. Technical content, various deformations or modifications are made within the scope of the claims, and other embodiments are easily made by means of replacement or change. Therefore, any changes and improvements made in the principles and process conditions of the present invention, etc. It should be included in the scope of the patent application of the present invention.

SEQUENCE LISTING

<110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences

<120> Construction method and application of MAP3K8 gene knockout PK-15 cell line

<130> None

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<170> PatentIn version 3.5

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Claims (7)

1. The PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out is characterized in that the preservation number of the PK-15 cell line PK-15-KO-MAP3K8 is CCTCC NO: C2019176.

2. The construction method of the PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out is characterized by comprising the following steps:

step 1: primer design and plasmid construction: respectively designing two sgRNAs according to a swine MAP3K8 gene sequence, and connecting double-stranded sg RNA with a digested lentiGuide-EGFP vector to obtain a recombinant lentivirus expression plasmid lentiGuide-EGFP-MAP3K8-sg RNA;

step 2: plasmid transfection: co-transfecting lentiGuide-EGFP-MAP3K8-sg RNA lentivirus expression plasmid and virus packaging auxiliary plasmid into PK-15 cells;

and step 3: and (3) slow virus concentration: collecting virus liquid, filtering, ultracentrifuging, concentrating and purifying virus, and storing for later use;

and 4, step 4: lentivirus infection and flow sorting: infecting PK-15 cells with lentiviruses with multiplicity of infection MOI =30, and then performing single cell sorting with an ultra-speed flow cytometric sorting system;

and 5: culturing and sequencing identification of monoclonal cells: and carrying out amplification culture and sequencing on the single cell obtained by sorting to identify the single cell clone.

3. The method for constructing a MAP3K8 gene knock-out PK-15 cell line PK-15-KO-MAP3K8 as claimed in claim 2, wherein the sequence of sgRNA is as follows:

P-MAP3K8-sgRNA1 Forward：5’-CACCGTTCCATAATGTCTATTACAT-3’，

P-MAP3K8-sgRNA1Reverse：5’-AAACATGTAATAGACATTATGGAAC-3’；

P-MAP3K8-sgRNA2Forward：5’-CACCGAGATCCCAGATTCCTGGGGT-3’，

P-MAP3K8-sgRNA2 Reverse: 5'-AAACACCCCAGGAATCTGGGATCTC-3', respectively; the lentiGuide-EGFP vector in step 1 was digested with BsmBI I enzyme and ligated to sg RNA.

4. The PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out is applied to the research of the action mechanism of virus infection.

5. The use of the MAP3K8 gene knock-out PK-15 cell line PK-15-KO-MAP3K8 of claim 4 for studying the mechanism of action of a viral infection, wherein said virus is FMDV or SVV.

6. Use of MAP3K8 gene knock-out PK-15 cell line PK-15-KO-MAP3K8 for isolating and culturing FMDV or SVV.

7. The PK-15 cell line PK-15-KO-MAP3K8 with the MAP3K8 gene knocked out is applied to large-scale cell culture and production of FMDV or SVV vaccine strains.

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