CN110129272A - PK-15 cell line stably expressing MAP3K8 protein and its construction and application - Google Patents

Abstract

The present invention relates to a PK-15 cell line stably expressing MAP3K8 protein and its construction and application. The cell line is named PK‑15/MAP3K8, and the deposit number is CCTCC NO: C2018262. The preparation of the cell line includes the following steps: synthesis of the target gene MAP3K8, construction of the recombinant lentiviral vector Lv-MAP3K8, packaging of the lentivirus expressing MAP3K8, infection of PK-15 cells with the lentivirus, screening, cultivation and identification to obtain stable expression of the MAP3K8 protein PK-15 cell line. Compared with wild-type PK-15 cells, the use of PK-15 cells to express a large amount of MAP3K8 can significantly inhibit the replication of FMDV or SVV. The establishment of this cell line provides biological materials for studying the function of MAP3K8 gene in porcine cells and the molecular mechanism behind it.

Description

PK-15 cell line stably expressing MAP3K8 protein and its construction and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a PK-15 cell line stably expressing MAP3K8 protein and its construction and application.

Background technique

Stable cell line means that after the plasmid is integrated into the chromosome, the cell line is screened with the resistance marker in the corresponding plasmid DNA, and a cell line with stable expression is obtained. The stable expression of exogenous DNA can be integrated into the chromosome of the host cell, or equivalent to the sustainable existence of the episomal, allowing the host cell to express the target gene for a long time. Lentivirus is a type of retrovirus, including a variety of mammalian immunodeficiency viruses. After the lentivirus infects the cells, the gene can be integrated into the chromosome of the host cell and expressed stably. Therefore, it is often transformed into a tool for genetic modification. Play an important role in disease treatment, transgenic animals, animal disease models, etc. Lentiviral vectors can accommodate larger DNA fragments and may infect a variety of dividing and non-dividing cells. The vector can effectively transfer the target gene into the brain, liver, muscle, retina, etc., without causing toxicity or immune response . In cell construction, the gene of interest can be inserted into the lentiviral vector, and the target cell can be infected with the lentivirus to integrate the gene of interest into the target cell, which is widely used in the construction of cell lines. With the continuous progress and development of animal transgenic technology, transgenic animals have broad application prospects in livestock improvement, medicine and hygiene, and biopharmaceuticals. As a type of vector derived from retroviruses, lentiviral vectors can integrate target genes into host cell chromosomes and express foreign genes stably for a long time. At the same time, they have high transfection efficiency, can infect dividing and non-dividing cells, It has the advantages of accommodating large exogenous gene fragments and is an efficient tool for preparing transgenic animals.

PK-15 cells (Porcine Kidney Epithelial cells), also known as PK15 or PK (15), are derived from pig kidneys. The Chinese name is pig kidney epithelial cells. The normal cell morphology is epithelial and grows adherently. 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, and swine fever virus Preparation of vaccines, etc. The proliferation ability of porcine kidney cell PK15 depends on comprehensive factors such as cell material, culture technology, and culture conditions. PK-15 cells have been widely used in the isolation, in vitro culture and production of related vaccines of classical swine fever virus, porcine pseudorabies virus and porcine parvovirus. So far, there are no reports on PK-15 cell lines that can restrict Foot-and-mouth disease virus (FMDV) and Seneca valley virus (SVV).

MAP3K8, also known as TPL2, is widely expressed in different tissues. This protein is a serine-threonine kinase. TPL2 signaling plays an important role in innate and adaptive immunity as well as in cancer as a potential proto-oncogene. The role of TPL2 in innate immunity is limited to bacterial infection models, and there are relatively few related studies using viruses and parasites. TPL2/MAP3K8 has been recognized as a key regulator of type I (IFN-α/β) and type II (IFN-γ) IFN, is an important component of multiple viral infection pathways, and differentially regulates IFN in a cell type-specific manner - Induction of α/β and IFN-λ. Studies have reported increased vesicular stomatitis virus (VSV) replication in TPL2-deficient mouse embryonic fibroblasts (Schmid, S.; Sachs, D. et al. Mitogen-activated protein kinase-mediatedlicensing of interferon regulatory factor 3/ 7 reinforces the cell response to virus. J Biol Chem 2014, 289 (1), 299-311.).

Contents of the invention

The purpose of the present invention is to provide a PK-15 cell line stably expressing MAP3K8 protein and its construction and application. The invention obtains the cell line PK-15/MAP3K8 stably expressing the MAP3K8 protein through the lentivirus expression system. The constructed cell line is infected with FMDV or SVV, and the cell line can inhibit the replication of FMDV and SVV. The establishment of this cell line laid the foundation for the isolation of virus and the study of the biological activity of MAP3K8.

The PK-15 cell line stably expressing MAP3K8 protein provided by the present invention is named PK-15/MAP3K8, and its deposit number is CCTCC NO: C2018261.

The invention also provides the application of PK-15 cell line in inhibiting FMDV or SVV replication.

The invention also provides the application of the PK-15 cell strain in the study of the gene function and molecular mechanism of MAP3K8 in pig-derived cells.

The present invention also provides a method for constructing a PK-15 cell line stably expressing MAP3K8 protein, which specifically includes the following steps:

Step 1: Construction of the recombinant lentiviral vector Lv-MAP3K8: insert the MAP3K8 gene into the MCS region of the lentiviral vector Lv-pCDH;

Step 2: Pack the lentivirus expressing MAP3K8: use 293FT cells to pack the Lv-MAP3K8 virus liquid; collect the virus liquid after 48 hours, filter and save it for later use;

Step 3: Infect PK-15 cells with lentivirus: Infect PK-15 cells with the lentivirus obtained in Step 2, and terminate after 8 hours;

Step 4: Screening and culturing: 48 hours after infection, drug screening was carried out. After 48 hours to 72 hours, the culture medium was replaced and passaged. The culture was maintained and identified, and the PK-15 cell line PK-15/MAP3K8 stably expressing MAP3K8 protein was obtained.

The present invention also uses qRT-PCR to verify the cell line PK-15/MAP3K8 screened through the above steps, and the results show that MAP3K8 can be efficiently and stably expressed in the cell line.

In the present invention, the PK-15/MAP3K8 cell line was respectively inoculated with FMDV and SVV, and the qRT-PCR results showed that, compared with wild-type PK-15, the cell line PK-15/MAP3K8 inhibited the replication of FMDV or SVV.

The present invention has the following beneficial effects:

1. The present invention uses a lentivirus system to construct a PK-15 cell line stably expressing MAP3K8 protein. The establishment of this cell line provides a reliable biological material for studying the function and molecular mechanism of the MAP3K8 gene in PK-15 cells.

2. The PK-15 cell line stably expressing the MAP3K8 protein constructed by the present invention can inhibit the replication of FMDV or SVV by using a large amount of PK-15 cells to express MAP3K8. and SVV, the relative expression of FMDV in wild-type PK-15 was higher than that in PK-15/MAP3K8 cells, and the difference was significant; the relative expression of SVV in wild-type PK-15 cells was higher than that in PK-15/MAP3K8 cells The relative expression levels of SVV in the two groups were significantly different. In conclusion, the constructed PK-15/MAP3K8 cell line has obvious antiviral effect and can significantly inhibit the replication of FMDV and SVV.

Description of drawings

Figure 1 is a graph comparing the expression levels of MAP3K8 in cell lines pCDH-PK-15 and PK-15/MAP3K8.

Fig. 2 is the electrophoresis detection diagram of qRT-PCR products of MAP3K8 and β-actin of cell lines pCDH-PK-15 and PK-15/MAP3K8.

Figure 3 is a graph showing dose-dependent inhibition of FMDV replication by MAP3K8. MYC-TPL2 represents the fusion expression product of MYC tag and MAP3K8.

Figure 4 is a comparison chart of MAP3K8 protein expression in wild-type PK-15 and PK-15/MAP3K8 cells after inoculation with FMDV, ** in the figure indicates p < 0.01, *** indicates p < 0.001.

Figure 5 is a comparison chart of FMDV/GAPDH mRNA expression in PK-15/MAP3K8 cells and wild-type PK-15, in the figure * indicates p < 0.05, ** indicates p < 0.01.

Fig. 6 is an indirect immunofluorescence observation photograph of PK-15/MAP3K8 cells and wild-type PK-15 after inoculation with FMDV for 16 hours. A: PK-15 infected with FMDV for 16h; B: PK-15/MAP3K8 infected with FMDV for 16h.

Figure 7 is a comparison chart of SVV/GAPDH mRNA expression in PK-15/MAP3K8 cells and wild-type PK-15, where ** in the figure indicates p < 0.01, and *** indicates p < 0.001.

Fig. 8 is an indirect immunofluorescence observation photograph of PK-15/MAP3K8 cells and wild-type PK-15 after inoculation with SVV for 16 hours. A: PK-15 infected with SVV 16h; B: PK-15/MAP3K8 infected with SVV 16h.

In the figures, MAP3K8-PK15 or MAP3K8(Pig)-PK15 represents PK-15/MAP3K8.

Preservation information:

Preservation time: December 19, 2018;

Name of depository unit: China Center for Type Culture Collection;

Deposit number: CCTCC NO: 2018262;

Address of Preservation Unit: No. 16, Luojiashan Street, Wuchang District, Wuhan City, Hubei Province, China;

Classification and name: pig-derived kidney cells PK-15/MAP3K8.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Sources of materials used in the examples: foot-and-mouth disease virus strain A/GDMM/CHA/2013, Seneca valley virus (SVV) strain and pig kidney cells (PK-15) were provided by the Lanzhou Veterinary Research Institute Foot-and-Mouth Disease Epidemic Scientific team; mouse anti-β-Actin monoclonal antibody was purchased from Thermo Scientific. Opti-MEM, Lipo 2000, 0.25% EDTA trypsin and newborn bovine serum (FBS) were purchased from Gibco; DMEM cell culture medium and PBS solution were purchased from Hyclone; ECL color reagent was purchased from Thermo Scientific; NP-40 The lysate and PMSF were purchased from Beyontian; the reverse transcription kit was produced by Novizan Biotech. The lentivirus Lv-pCDH was purchased from the plasmid vector strain protein antibody cell gene collection center (NTCC). The recombinant lentiviral vector Lv-MAP3K8 was constructed by PPL (Public Protein/Plasmid Library, China). The VP3 protein rabbit primary antibody of FMDV and the rabbit primary antibody of SVV were prepared by the foot-and-mouth disease epidemiology team of Lanzhou Veterinary Research Institute. The preparation method refers to (Chen Fei. Preparation and identification of polyclonal antibody to 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 antibody with purified vesicular stomatitis virus antigen for teaching [J], Health Vocational Education, 2019 (6): 112-114). Immunofluorescence rabbit secondary antibody was purchased from Cell Signaling Technology (CST). Rabbit secondary antibodies were purchased from Proteintech Group.

Construction process of embodiment 1PK-15/MAP3K8 stable cell line

1.1 Construction of lentiviral vector and virus packaging

Specifically include the following steps:

Step 1: Construction of recombinant lentiviral vectors Lv-MAP3K8 and Lv-pCDH: the CDS sequence of the Susscrofa (pig) MAP3K8 gene by chemical synthesis, see XM_021064737.1 on NCBI for the CDS sequence; the 5' end and 3' end of the sequence are introduced into The restriction sites Xba I and BamH I were cut, and the target gene and the lentiviral vector Lv-pCDH vector were double-digested and reconnected to obtain the recombinant lentiviral vector Lv-MAP3K8.

Step 2: Packaging lentivirus:

(1) 293FT cells were used to package the lentivirus Lv-pCDH and Lv-MAP3K8 virus liquid respectively;

① Digestion and inoculation of 293T cells: Digest well-growing 293T cells with 0.25% trypsin to prepare a single cell suspension and adjust the concentration to 4×10 ⁵ /ml, and inoculate 10 ml into a 10 cm culture dish. Cultivate overnight in a 37˚C, 5% CO ₂ incubator, and the next day the cells reach 80% confluency, and set aside.

②Virus packaging:

Virus packaging: 7.5 µg lentiviral vector Lv-pCDH and packaging plasmid (3 µg pMD2G and 6 µg psPAX), 7.5 µg recombinant lentiviral vector Lv-MAP3K8 (hamster) and packaging plasmid (3 µg pMD2G and 6 µg psPAX), respectively Add it to 150 µl OPTI-MEM and mix well to get two sets of lentiviral vector mixture; take 25 µl Lipo 2000 and add it to 500 µl OPTI-MEM and mix well, let stand at room temperature for 5 min to get Lipo 2000 mixture; mix two sets of lentiviral vector The mixture was slowly added to the Lipo 2000 mixture, mixed well and then allowed to stand at room temperature for 15 min. Then add them drop by drop to the 293T cells obtained in step ① with a confluence of 80%, and mix well. After 6 h, the medium was replaced with fresh DMEM containing 10% FBS.

Requirements for lentiviral vector: the concentration is 1 µg/µl, the purity: the value of OD260/OD280 is in the range of 1.8-2.0.

(2) After culturing for 48 hours, collect the two groups of culture fluids (including lentivirus), centrifuge at 1500g, 4 ˚C for 10 minutes to remove cell debris, then filter the virus supernatant with a 0.45 μm filter membrane and filter with a 0.45 μm filter , Save for later use.

1.2 Screening and identification of PK-15 cell line PK-15/MAP3K8 stably expressing MAP3K8

Specifically include the following:

1.2.1 Lentivirus infection of PK-15 cells:

Add the Lv-pCDH and Lv-MAP3K8 virus filtrate obtained in 1.1 to 5 × pEGit virus concentrate at a ratio of 4:1. After standing overnight at 4°C, centrifuge at 3200g, 4°C for 20 min. Discard the supernatant, resuspend the virus pellet in DMEM medium containing 5% FBS, aliquot 200 µl/tube, and store at -80 °C.

Then infect PK-15 cells with lentiviruses Lv-pCDH and Lv-MAP3K8 respectively: the volume of virus is 200 μl, the number of viruses is about 2×10 ⁷ , supplemented to 2 ml with complete medium, and 2 μl transfection enhancer polybrene (polybrene) is added , terminated after 8h. The two groups of cells were designated as pCDH-PK-15 and PK-15/MAP3K8 respectively.

1.2.2 Screening of pCDH-PK-15 and PK-15/MAP3K8 cell lines:

After 48 hours of infection, puromycin was added for drug screening. After 48 hours to 72 hours of screening, the medium was replaced and passaged, and the culture was maintained to obtain positive cell lines pCDH-PK-15 and PK-15/MAP3K8. The positive cell line PK-15/MAP3K8 obtained by screening is the PK-15 cell line stably expressing MAP3K8 protein.

1.2.3 qRT-PCR detection of the expression level of MAP3K8:

(1) Design qRT-PCR primers for MAP3K8 (pig) and qRT-PCR primers for β-actin gene, the primer sequences are shown in Table 1 (SEQ ID NO: 1-4).

(2) Trizol extracted RNA from positive pCDH-PK-15 and PK-15/MAP3K8 cell lines, and determined the concentration of RNA.

(3) Perform reverse transcription according to the standard of reverse transcription of 500ng RNA into 10µl system cDNA, use the successfully reverse-transcribed cDNA as a template, and use the primers synthesized in step 1 as amplification primers for qRT-PCR detection. The reaction system is: AceQ®qPCRSYBR®Green Master Mix 10µl, sterilized ddH ₂ O 7.2µl, Primer1 (10µM) 0.4µl, Primer2 (10µM) 0.4µl, template cDNA 2 µl. The amplification program was: 95°C for 5min; 95°C for 10s, 60°C for 30s, 40 cycles; 95°C for 15s, 60°C for 60s, 95°C for 15s. The qRT-PCR results showed that the expression of MAP3K8 in the PK-15/MAP3K8 cell line at the mRNA level was about 250 times higher than that of the control cell line pCDH-PK-15.

(4) After the qRT-PCR experiment, the nucleic acid electrophoresis technique was used to detect the qRT-PCR product. The results showed that: for the MAP3K8 gene, the band of the qRT-PCR amplification product of the PK-15/MAP3K8 cell line was similar to that of pCDH- Compared with the PK-15 cell lines, the difference is significant; for the β-actin gene, the bands of the qRT-PCR amplification products of the two cell lines are basically the same.

The results of qRT-PCR detection and electrophoresis showed that the cell line PK-15 stably expressing MAP3K8 was screened through the above steps, and MAP3K8 could be expressed efficiently and stably in this cell line.

Table 1 Real-time quantitative PCR primers Primer name sequence (5'-3') serial number MAP3K8-pig-F TCGACCAAAGCCGACATCTA SEQ ID NO:1 MAP3K8-pig-R TATCAGCTCCCTCATGCCTG SEQ ID NO:2 β-actin-pig-F TCCCTGGAGAAGAGCTACGA SEQ ID NO:3 β-actin-pig-R CGCACTTCATGATCGAGTTG SEQ ID NO:4 GAPDH-F ACATGGCCTCCAAGGAGTAAGA SEQ ID NO:5 GAPDH-R GATCGAGTTGGGGCTGTGACT SEQ ID NO:6 FMDV-F CACTGGTGACAGGCTAAGG SEQ ID NO:7 FMDV-R CCTTTCCAGATTCCGAGT SEQ ID NO:8 SVV-F AGAATTTGGAAGCCATGCTCT SEQ ID NO:9 SVV-R GAGCCAACATAGAAACAGATTGC SEQ ID NO:10

Example 2

2.1 MAP3K8 dose-dependent inhibition of FMDV replication

(1) Preparation of pCDNA3.1-MAP3K8(Pig):

pCDNA3.1-MAP3K8 (Pig) was synthesized by Shanghai Jinkairui Company, specifically: a BamHI restriction site and a MYC tag were attached to the 5' end of the MAP3K8 (Pig) sequence, and an XhoI restriction site was attached to the 3' end, and then The sequence and the pCDNA3.1(+) vector were digested with BamHI and XhoI respectively, and ligated to obtain the recombinant pCDNA3.1-MAP3K8(Pig).

MAP3K8 (Pig) sequence information: refer to the CDS sequence shown in NCBI serial number XM_013980209.2.

(2) PK-15 cells were dose-dependently transfected with pCDNA3.1-MAP3K8(Pig):

0μg, 1μg and 2μg pCDNA3.1-MAP3K8(Pig) were transfected into 3 groups of the same PK-15 cells (cell confluency reached 80%), and then inoculated with FMDV 24 hours later at a dose of 1:200. Collect the samples 6 hours after inoculation.

(3) Preparation of PK-15 cell protein samples:

Discard the culture medium, rinse the cells twice with 1×PBS to remove cell debris; add an appropriate amount of NP-40 lysate, and add PMSF within a few minutes before use, so that the final concentration of PMSF is 1 mol·L ^-1 ; After fully lysed, centrifuge at 12,000 r min ^-1 for 10 min, collect the supernatant of the sample in a newly prepared EP tube, add 5×SDS Loading Buffer (beta-mercaptoethanol has been added), and cook the sample in a metal bath at 100°C for 10 minutes. ~15 min.

(4) SDS-PAGE protein gel electrophoresis and transfer:

Prepare 10% polyacrylamide protein gel; take an appropriate amount of protein sample and add a protein pre-stained marker as an indicator of the size; first adjust the electrophoresis voltage to 80 V and run for 30 minutes, after the protein sample runs out of the stacking gel and enters the separating gel , adjust the voltage to 120 V until the end of the electrophoresis; after the end of the electrophoresis, 200 mA, transfer the membrane for 2 hours; after the end of the transfer, use 5% skimmed milk powder to block at room temperature for 2 hours; after blocking, wash twice with TBST, each time for 5 minutes ; Incubate primary antibody overnight at 4°C (primary antibody can be recycled); TBST quick wash 3 times, 10 min each; incubate secondary antibody, room temperature for 2 h; TBST quick wash 3 times, 10 min each; The image acquisition system performs ECL development and saves the results. The primary protein antibody of FMDV is VP3 protein rabbit primary antibody, the primary antibody of MYC tag was purchased from Sigma Company, and the primary antibody of β-actin, rabbit secondary antibody and mouse secondary antibody were purchased from Proteintech Group Company.

As shown in Figure 3, with the increase of the amount of pCDNA3.1-MAP3K8 (Pig) transfection, the protein band of FMDV gradually faded, indicating that MAP3K8 dose-dependently inhibited the replication of FMDV, and MAP3K8 has the ability to make the host anti-virus, natural immunity.

2.2qRT-PCR verification that PK-15/MAP3K8 cells inhibit FMDV replication

Include the following steps:

(1) Dilute FMDV venom to a concentration of 1:500 with serum-free DMEM.

(2) Digest PK-15/MAP3K8 cells and place them on cell plates, place them in a 37°C, 5% CO ₂ incubator, and wash the cells with serum-free DMEM when the cells grow to 70%-90% Two times to remove residual serum from the cells.

(3) Add the diluted venom to the cells according to an appropriate volume (2ml of cell culture medium plus 4ul of FMDV), incubate in a 37°C, 5% CO ₂ incubator for 1 hour, discard the venom, and replace with 2% FBS DMEM maintenance solution, continue to culture. Samples were collected at 0, 8h, and 16h after inoculation.

(4) Extract sample RNA, reverse transcribe, and then use the primer pair MAP3K8-pig-F/R (SEQ ID NO: 1-2) to amplify the MAP3K8 gene and the primer pair FMDV-F/R to amplify the 3D protein of FMDV R (SEQ ID NO: 7-8) and the primer pair GAPDH-F/R (SEQ ID NO: 5-6) for amplifying the internal reference GAPDH gene were used to perform qRT-PCR on the reverse-transcribed cDNA respectively. For the sequence of the primer pair, see Table 1.

As shown in Figure 4, in the samples collected at 0h, 8h, and 16h after inoculation, the expression level of MAP3K8 in PK-15/MAP3K8 cells was higher than that in control wild-type PK-15 cells (the difference was extremely significant). This indicated that PK-15/MAP3K8 cells overexpressed MAP3K8 under FMDV stimulation.

As shown in Figure 5, in the samples collected at 8h and 16h after inoculation, the expression of FMDV/GAPDH mRNA in PK-15/MAP3K8 cells was lower than that in wild-type PK-15 cells (significant difference) . That is to say, PK-15/MAP3K8 cells can inhibit the replication of FMDV at 8h and 16h after inoculation compared with wild-type PK-15 cells. This is because PK-15/MAP3K8 cells overexpress MAP3K8 under FMDV stimulation, thereby inhibiting the replication of FMDV. This indicates that PK-15/MAP3K8 cells can be used to overexpress MAP3K8 to inhibit the replication of FMDV.

2.3 Indirect immunofluorescence verification that PK-15/MAP3K8 cells inhibit FMDV replication

(1) Spread PK-15 cells in a 20 mm glass dish, and inoculate the cells when the cells grow to 40%-50%;

(2) Harvest cells 16 hours after inoculation and wash once with 1×PBS;

(3) Fix with 4% paraformaldehyde (1 mL per dish) at room temperature in the dark for 1 h;

(4) Wash 3 times with 1×PBS, 5 min/time (add liquid lightly to prevent cells from being washed up);

(5) Permeabilize with 0.2% Triton X-100 (100 mL PBS+20 μL Triton100) at room temperature for 1 hour;

(6) Wash 3 times with 1×PBS, 5 min each time;

(7) Block with 5% BSA (1×PBST: 50 mL 1×PBS+50 μL Tween20, 5% BSA: 10 mL +0.5g BSA) at 37°C for 1 h;

(8) Aspirate the blocking solution, add the primary antibody (FMDV VP3 protein rabbit primary antibody) diluted with 5% BSA, and overnight at 4 °C;

(9) Wash three times with 1×PBST, 10 min each time, add fluorescein-labeled secondary antibody (immunofluorescence rabbit secondary antibody) diluted in 1×PBST (protect from light), incubate at 37°C for 1 h;

(10) Wash three times with 1×PBST, 10 min/time, add 100 μL of reduced-sealing tablet (containing DAPI) to each dish;

(11) Use laser confocal instrument to observe the fluorescence and save the picture.

The results are shown in Figure 6, in the samples collected 16 hours after inoculation, the degree of lesion of PK-15/MAP3K8 cells was much lower than that of wild-type PK-15 cells. That is to say, PK-15/MAP3K8 cells inhibited the replication of FMDV at 16h compared with wild-type PK-15. This is because PK-15/MAP3K8 cells overexpress MAP3K8 under FMDV stimulation, thereby inhibiting the replication of FMDV. This indicates that PK-15/MAP3K8 cells can be used to overexpress MAP3K8 to inhibit the replication of FMDV.

2.4 Determination of TCID ₅₀ of FMDV in cell line PK-15/MAP3K8

FMDV was serially diluted ^10-1 to ^10-8 times, and inoculated into 96-well cell culture plates filled with monolayers of PK-15 cells and PK-15/MAP3K8 cells, respectively, inoculating 8 wells for each dilution, 0.1 mL/ Wells were cultured in an incubator at 37°C for 5-7 days, the cytopathic changes (CPE) were observed and recorded daily, and the TCID ₅₀ of the isolated virus was calculated according to the Reed-Munch method. The TCID ₅₀ of FMDV was determined by Reed-Muench method, the TCID ₅₀ of PK-15 cells was 10 ^-5.8 /mL, and the TCID ₅₀ of PK-15/MAP3K8 cell line was 10 ^-4.4 /mL. This indicated that PK-15/MAP3K8 cells inhibited the replication of FMDV compared with wild-type PK-15.

Example 3

3.1qRT-PCR verification that PK-15/MAP3K8 cells inhibit SVV replication

Include the following steps:

(1) Dilute SVV venom to a concentration of 1:500 with serum-free DMEM.

(2) Digest PK-15/MAP3K8 cells and place them on cell plates, place them in a 37°C, 5% CO ₂ incubator, and wash the cells with serum-free DMEM when the cells grow to 70%-90% Two times to remove residual serum from the cells.

(3) Add the diluted venom to the cells according to an appropriate volume (2ml of cell culture medium plus 4ul of FMDV), incubate in a 37°C, 5% CO ₂ incubator for 1 hour, discard the venom, and replace with 2% FBS DMEM maintenance solution, continue to culture. Samples were collected at 0, 8h, and 16h after inoculation.

(4) Extract the sample RNA, reverse transcribe it into cDNA, and then perform qRT-PCR to amplify the primer pair of the 3D protein conserved region of SVV (SEQ ID NO:9~10) and the primer pair for amplifying the internal reference GAPDH gene (SEQ ID NO:9-10) ID NO:5~6) See Table 1 for details.

The results are shown in Figure 7. In the samples collected at 8h and 16h after inoculation, the expression of SVV/GAPDH mRNA in PK-15/MAP3K8 cells was lower than that in wild-type PK-15 cells (difference significant). That is to say, PK-15/MAP3K8 cells can inhibit the replication of SVV at 8h and 16h after inoculation compared with wild-type PK-15. This is because PK-15/MAP3K8 cells overexpress MAP3K8 under SVV stimulation, thereby inhibiting the replication of SVV. This suggests that PK-15/MAP3K8 cells can be used to overexpress MAP3K8 to inhibit SVV replication.

3.2 Indirect immunofluorescence verification that PK-15/MAP3K8 cells inhibit SVV replication

(1) Spread PK-15 cells in a 20 mm glass dish, and inoculate the cells when the cells grow to 40%-50%;

(2) Harvest cells 16 hours after inoculation and wash once with 1×PBS;

(3) Fix with 4% paraformaldehyde (1 mL per dish) at room temperature in the dark for 1 h;

(4) Wash 3 times with 1×PBS, 5 min/time (lightly add to prevent cells from being washed up);

(5) Permeabilize with 0.2% Triton X-100 (100 mL PBS+20 μL Triton100) at room temperature for 1 hour;

(6) Wash 3 times with 1×PBS, 5 min each time;

(7) Block with 5% BSA (1×PBST: 50 mL 1×PBS+50 μL Tween20, 5% BSA: 10 mL +0.5g BSA) at 37°C for 1 h;

(8) Aspirate the blocking solution, add the primary antibody (primary antibody to SVV rabbit) diluted with 5% BSA, and store overnight at 4°C;

(9) Wash three times with 1×PBST, 10 min each time, add fluorescein-labeled secondary antibody (immunofluorescence rabbit secondary antibody) diluted in 1×PBST (protect from light), incubate at 37°C for 1 h;

(10) Wash three times with 1×PBST, 10 min/time, add 100 μL of reduced-sealing tablet (containing DAPI) to each dish;

(11) Use laser confocal instrument to observe the fluorescence and save the picture.

The results are shown in Figure 8, in the samples collected 16 hours after inoculation, the degree of lesion of PK-15/MAP3K8 cells was much lower than that of wild-type PK-15 cells. It shows that PK-15/MAP3K8 cells can inhibit the replication of SVV at 16h compared with wild-type PK-15. This is because PK-15/MAP3K8 cells overexpress MAP3K8 under SVV stimulation, thereby inhibiting the replication of SVV. This suggests that PK-15/MAP3K8 cells can be used to overexpress MAP3K8 to inhibit SVV replication.

3.3 Determination of TCID ₅₀ of SVV in cell line PK-15/MAP3K8

SVV was serially diluted ^10-1 to ^10-8 times, and inoculated into 96-well cell culture plates filled with monolayer PK-15 cells and PK-15/MAP3K8 cells, respectively, inoculating 8 wells for each dilution, 0.1 mL/ Wells were cultured in an incubator at 37°C for 5-7 days, the cytopathic changes (CPE) were observed and recorded daily, and the TCID ₅₀ of the isolated virus was calculated according to the Reed-Munch method. The TCID ₅₀ of SVV was determined by Reed-Muench method, and the TCID ₅₀ of PK-15 cells was 10 ^-8.5 /mL. The TCID ₅₀ assay result of PK-15/MAP3K8 cell line was 10 ^-5.5 /mL. This indicated that PK-15/MAP3K8 cells inhibited the replication of SVV compared to wild-type PK-15.

The embodiments described above are only preferred embodiments of the present invention, and are only used to explain the present invention, not to limit the implementation scope of the present invention. Technical content, other implementation modes can be easily made through replacement or change, so all changes and improvements made in the principles and process conditions of the present invention should be included in the patent scope of the present invention.

SEQUENCE LISTING

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

<120> PK-15 cell line stably expressing MAP3K8 protein and its construction and application

<130> None

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tcgaccaaag ccgacatcta 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tatcagctcc ctcatgcctg 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tccctggaga agagctacga 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cgcacttcat gatcgagttg 20

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

acatggcctc caaggagtaa ga 22

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gatcgagttg gggctgtgac t 21

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cactggtgac aggctaagg 19

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cccttctcag attccgagt 19

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agaatttgga agccatgctc t 21

<210> 10

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gagccaacat agaaacagat tgc 23

Claims (4)

1. stablizing the PK-15 cell strain of expression MAP3K8 albumen, which is characterized in that the cell strain is named as PK-15/MAP3K8, Deposit number is CCTCC NO:C2018262.

2. PK-15 cell strain described in claim 1 is inhibiting the application in FMDV or SVV duplication.

3. PK-15 cell strain described in claim 1 is in pig source cellMAP3K8Gene function and its molecular mechanism grind Application in studying carefully.

4. stablizing the construction method of the PK-15 cell strain of expression MAP3K8 albumen, which is characterized in that specifically includes the following steps:

Step 1: MAP3K8 gene the building of recombined lentivirus vector Lv-MAP3K8: being inserted into slow virus carrier Lv-pCDH's The area MCS；

Step 2: the slow virus of packaging expression MAP3K8: packing Lv-MAP3K8 virus liquid with 293FT cell；It is collected after 48 hours Virus liquid is filtered, is saved backup；

Step 3: slow-virus infection PK-15 cell: the slow-virus infection PK-15 cell that step 2 is obtained terminates after 8 hours；

Step 4: screening and culturing: after infection 48h, carrying out drug screening, culture medium is replaced after 48h~72h and is passed on, training is maintained It supports, identification, obtains the PK-15 cell strain PK-15/MAP3K8 for stablizing expression MAP3K8 albumen.