CN111549059A - TPL2 gene knockout HEK293T cell line and its construction method and application - Google Patents

Abstract

The invention relates to a TPL 2gene knockout HEK293T cell line and a construction method and application thereof. The invention provides a construction method of a TPL 2gene knockout HEK293T cell line, which takes a second exon region of a human TPL 2gene as a target sequence, and concretely takes a 390 th to 459 th bp sequence or a 391 th to 448 th bp sequence of a TPL2 allele 1 and a second exon of an allele 2 of SEQ ID NO:1 as the target sequence. The invention provides a TPL 2gene knockout HEK293T cell line HEK293T-KO-TPL2 with a preservation number of CCTCC NO: C2019328. The morphology, proliferation speed and other aspects of the knockout cell line obtained by the invention have no obvious difference from those of a control cell, and the knockout cell line is an ideal TPL2 knockout cell model; the modified cell line is stable, provides a key biological material for researching the mode of inhibiting virus replication by TPL2 protein and the pathogenic mechanism of the virus, and can be used for separating and culturing SVA and applied to large-scale cell culture and production of SVA vaccine strains.

Description

TPL2 gene knockout HEK293T cell line and its construction method and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a TPL2 gene knockout HEK293T cell line and a construction method and application thereof.

Background technique

The HEK293 cell line is a human embryonic kidney epithelial cell transfected with the adenovirus E1A gene. HEK293T cells are highly transduced strains formed by transferring SV40T-antigen gene into HEK293 cells, which can express the SV40 large T antigen, and the plasmids containing the SV40 origin of replication and promoter region can replicate. In addition to various gene expression and protein production, the cells can also be used for the production of high-titer retroviruses and other viruses, such as adenoviruses and other mammalian viruses.

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, SRISKANTHARAJAH S, LEY S C. Regulation and function of TPL-2, an IkappaB kinase-regulated MAP kinasekinasekinase [J]. Cell Res, 2011, 21(1): 131-45.). After stimulation of various receptors such as TLR, TNFR and IL1R, they can regulate the activation of downstream signaling proteins such as ERK, JNK, p38 and NF-κB through TPL2-mediated signal transduction; A variety of innate immune cells such as dendritic cells and neutrophils produce a large number of cytokines such as type I interferon and tumor necrosis factor (GANTKE T, SRISKANTHARAJAH S, SADOWSKI M, et al. IκB kinase regulation of the TPL-2 /ERK MAPK pathway. [J]. Immunol Rev, 2012, 246(1): 168-82.). TPL2 is also essential in regulating the differentiation of CD4+ T cells into different Th cell lineages (ZHU J, PAUL W E. CD4 T cells: fates, functions, and faults [J]. Blood, 2008, 112(5 ): 1557-69.), is an important player in innate immunity, inflammation and tumor, and plays an important role in both innate immunity and acquired immunity.

CRISPR/Cas9 gene editing technology is the third-generation genome editing technology developed rapidly after ZFN and TALEN technology. This 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. CRISPR/Cas9 genome editing technology specifically recognizes the target gene sequence through a segment of gRNA, and guides 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). Genomic DNA at breaks and introducing insertion or deletion mutations (CONG L, F Z. Genome engineering using CRISPR/Cas9system [J]. Methods MolBio, 2015, 1239: 197-217.). At present, three gene editing endonucleases, ZNF, TALEN and CRISPR/Cas9, have been used in clinical practice. Among them, the CRISPR/Cas9 system has become the most widely used gene editing technology in this field due to its advantages of high efficiency, rapidity, versatility, ease of use, and low cost, and has been applied in various species (MEMI F, NTOKOU A, PAPANGELII. CRISPR/Cas9 gene-editing: Research technologies, clinical applications and ethical considerations [J]. SeminPerinatol, 2018, 42(8): 487-500.).

The Chinese invention patent publication number CN 110862968 A discloses the MAP3K8 gene knockout PK-15 cell line PK-15-KO-MAP3K8 and its construction method and application. The cell line can promote the proliferation of FMDV and SVV, improve the virus yield, can be used for large-scale cellular culture and production of FMDV and SVV vaccine strains, and can provide a powerful tool for studying the mechanism of MAP3K8 in virus infection. However, in the later practice, it was found that the cell line was not stable enough compared with the wild cell line in terms of cell morphology and proliferation rate, which was not conducive to the application in basic research.

SUMMARY OF THE INVENTION

The present invention provides a TPL2 gene knockout HEK293T cell line HEK293T-KO-TPL2 in order to solve the problem of poor stability in cell shape and proliferation rate of the TPL2 gene knockout cell line. The growth rate, cell morphology and proliferation rate of the cell line HEK293T-KO-TPL2 were no different from those of the wild HEK293T cell line, which is an ideal TPL2 knockout cell model. Mechanism of accumulation of key biomaterials.

The present invention specifically adopts the following technical solutions:

In the first aspect, the present invention provides a method for constructing a TPL2 gene knockout HEK293T cell line, using the second exon region of the human TPL2 gene as the target sequence, specifically, the TPL2 allele 1 shown in SEQ ID NO: 1 is used as the target sequence. and the 390-459 bp sequence or the 391-448 bp sequence of the second exon of allele 2 as the target sequence.

As a preferred solution, the foreign sequence is knocked in or the target sequence is knocked out in the target sequences of allele 1 and allele 2.

As a preferred solution, an exogenous sequence is inserted between the 390-391 bp of the TPL2 allele 1 shown in SEQ ID NO: 1, and the base "G" at the 449 bp is knocked out, and the allele 2 is knocked out. 391~448bp total 58bp;

Alternatively, the "C" base of the 393 bp of the TPL2 allele 1 shown in SEQ ID NO: 1 is knocked out, and the 442-459 bp is knocked out for a total of 18 bp, and the 391-448 bp for the knock-out allele 2 is a total of 58 bp.

More preferably, the exogenous sequence inserted between the 390-391 bp of TPL2 allele 1 is shown in SEQ ID NO:6.

The construction method of the above-mentioned TPL2 gene knockout HEK293T cell line specifically includes the following steps:

Step 1: Design of sgRNA oligo sequence: construct two pairs of sgRNAs specifically targeting human TPL2 gene: sgRNA1 and sgRNA2 according to the sequence of human TPL2 gene;

Specifically, the sgRNA targets the second exon region of the human TPL2 gene.

Further, the sgRNA1 is synthesized from the following sequence:

H-TPL2-sgRNA1Forward: 5'-TCCTCGGGGCGCCTTTGGAA-3';

H-TPL2-sgRNA1Reverse: 5'-TTCCAAAGGCCGCCCCGAGGA-3';

The sgRNA2 was synthesized from the following sequence:

H-TPL2-sgRNA2Forward: 5'-CCGATGTTCTCCTGATCCCC-3';

H-TPL2-sgRNA2Reverse: 5'-GGGGATCAGGAGAACATCGG-3';

Step 2: Construction of pX-EZ-TPL2-sgRNA recombinant plasmid: The two sgRNAs constructed to specifically target and knock out the human TPL2 gene were cloned into the same CRISPR/Cas9 vector plasmid pX459 to obtain the recombinant plasmid pX-EZ-TPL2 -sgRNA;

Specifically, the EZ-GuideXH helper plasmid was used to clone the two sgRNAs into the same CRISPR/Cas9 vector plasmid pX459.

Step 3: Plasmid transfection: The pX-EZ-TPL2-sgRNA recombinant plasmid and the pX-EZ empty plasmid were respectively transfected into HEK293T cells;

Specifically, the transfection process is performed according to the instructions of HighGene transfection reagent.

Before transfection, HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

Step 4: Drug screening of monoclonal cell lines: use the CRISPR/Cas9 system to achieve gene silencing, kill negative cells through drug screening, and then obtain positive monoclonal cell lines by limiting dilution;

Specifically, the screening drug is a puromycin antibiotic.

Step 5: Identification of gene knockout cell lines: The sorted monoclonal cell lines were expanded and cultured, and the gene knockout status of positive monoclonal cell lines was first identified by genetyping PCR sequencing. The knockout cell lines were combined for further identification to verify the knockout of TPL2 protein in the cell lines.

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 then expanding the culture to a 24-well plate, a 12-well plate, and a 6-well plate in turn. and T25 flasks.

Further, the genetyping PCR detection primer sequence is:

H-TPL2 genetyping Forward: 5'-GACCAGGCACCTGCATCTGTT-3',

H-TPL2 genetyping Reverse: 5'-TGAGGCAGTGCACCCTCAGA-3'.

In a second aspect, the present invention provides a cell line constructed by the above-mentioned TPL2 gene knockout HEK293T cell line construction method.

Further, the present invention provides a TPL2 gene knockout HEK293T cell line HEK293T-KO-TPL2, whose deposit number is CCTCC NO: C2019328.

In a third aspect, the present invention provides the application of the TPL2 gene knockout HEK293T cell line in the study of the way that TPL2 inhibits virus replication and the pathogenic mechanism of the virus.

Further, the virus is SVA.

In a fourth aspect, the present invention provides the application of the TPL2 gene knockout HEK293T cell line in the isolation and culture of SVA.

In the fifth aspect, the present invention provides the application of the TPL2 gene knockout HEK293T cell line in the large-scale cellular culture and production of SVA vaccine strains.

The present invention has the following beneficial effects:

1. The present invention uses the CRISPR/Cas9 system to construct the TPL2 gene knockout HEK293T cell line HEK293T-KO-TPL2. After the transformation, the cell line is stable, and there is no significant difference in cell morphology, proliferation rate, etc. from the control cells. An ideal TPL2 knockout cell model provides key biological materials for exploring the way TPL2 protein inhibits virus replication and the pathogenic mechanism of the virus.

2. The HEK293T-KO-TPL2 cell line HEK293T-KO-TPL2 is knocked out of TPL2 gene, because the deletion of some fragments changes the open reading frame of the TPL2-encoded protein, resulting in frameshift mutation, so the TPL2 protein cannot be expressed correctly, and then the purpose of gene knockout is achieved. Due to the antiviral effect of TPL2 protein, the proliferation of SVA virus in wild-type HEK293T cells will be inhibited, and the TPL2 gene knockout HEK293T cell line HEK293T-KO-TPL2 cannot correctly express TPL2 protein, so it is conducive to the development of SVA virus in HEK293T cells. Proliferation in this cell line yielded a larger amount of high titer virus, proving that CRISPR/Cas9 gene editing technology to edit an engineered cell line for vaccine production is a feasible strategy to increase virus yield, and it is a promising strategy for large-scale cell lines of future SVA vaccine strains. Chemical culture and production are of great significance.

Description of drawings

FIG. 1 is a diagram showing the results of sequencing verification of the pX459-sgRNA1 and EZ-sgRNA2 recombinant plasmids constructed in the examples of the present invention.

Figure 2 is a diagram showing the results of PCR identification of the pX-EZ-TPL2-sgRNA recombinant plasmid constructed in the embodiment of the present invention, and the first to fourth lanes in the figure are Marker DL2000, recombinant plasmid pX-EZ-TPL2-sgRNA, blank lane, recombinant Plasmid pX-EZ-TPL2-sgRNA.

Figure 3 is a graph showing the results of genomic PCR identification of TPL2 knockout cell lines in the example of the present invention, lane 1 is Marker DL2000, lane 2 is HEK293T-WT-TPL2 cell line, and lane 3 is HEK293T-KO-TPL2-A2 cell line, Lane 5 is HEK293T-KO-TPL2-B1 cell line, other lanes are non-positive clones.

FIG. 4 is a diagram showing the sequencing results of the genotype comparison of the TPL2 knockout cell lines HEK293T-KO-TPL2-A2 and HEK293T-KO-TPL2-B1 in the example of the present invention.

Figure 5 is a graph showing the abundance of TPL2 protein in HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells detected by Western blotting in the example of the present invention.

FIG. 6 is a cell morphological diagram of HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells under an inverted microscope in the example of the present invention.

FIG. 7 is a time chart of the formation of cell monolayers by HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells in the example of the present invention.

Figure 8 shows the fluorescence expression of SVA in HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells observed under a fluorescence microscope in the example of the present invention.

Fig. 9 is a graph showing the absolute quantitative detection of the viral copy number of SVA replication in HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells in the embodiment of the present invention.

Figure 10 is a graph showing the relative quantitative detection of viral transcript levels of SVA replication in HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells in the example of the present invention.

Figure 11 is a graph showing the abundance of viral structural proteins detected by Western blotting in the HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells replicated by SVA.

Figure 12 is a graph of the SVATCID ₅₀ titer of HEK293T-WT-TPL2 and HEK293T-KO-TPL2 cells expanded in the examples of the present invention.

In the figures, *, p < 0.05 indicates a statistically significant difference; **, p < 0.01 indicates a very significant statistical difference.

Deposit information:

Preservation time: November 29, 2019;

Name of the depositary unit: China Type Culture Collection;

Deposit number: CCTCC NO: C2019328;

Depositary address: Wuhan University, China;

Classification name: Human embryonic kidney cells HEK293T-KO-TPL2.

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, plasmids and viruses: Human embryonic kidney 293T cells (HEK293T) were provided by ABclonal. The CRISPR/Cas9 vector plasmid pX459 pSpCas9-2A-puro-MCS and the helper vector plasmid EZ-GuideXH were provided by ABclonal Company. Escherichia coli Trans5α competent cells were purchased from Quanzhou Gold Company. Seneca Valley virus (SVA) strains were preserved by the foot-and-mouth disease epidemiology team of Lanzhou Veterinary Research Institute.

Reagents and antibodies: 0.25% trypsin, Opti-MEM, DMEM medium, penicillin and heat-inactivated fetal bovine serum (FBS) were purchased from Gibco; T4 DNA ligase (Quick), HighGene transfection reagent were Purchased from ABclonal Company; SDS-PAGE protein loading buffer (5X), RNA extraction reagent Trizol, 5×First buffer, 0.1M DTT, RNase inhibitor (RRI) and reverse transcriptase M-MLV were purchased from Invitrogen Company; Oligo(dT) primers, Random primers, deoxyribonucleoside triphosphates (dNTPs), 2×one step RT-PCR Buffer III, TaKaPa EX Taq HS and PrimerScript RT Enzyme mix II were purchased from Takara Company; Phosphate Salt buffer (PBS solution pH7.4, 0.0067M) was purchased from Hyclone Company; RAPI cell lysate and PMSF were purchased from Biyuntian Company; restriction endonucleases Bbs1, Spe1, Kpn1, protein prestained Marker, ECL display Color solutions were purchased from Thermo Fisher Scientific; nitrocellulose membrane (NC membrane) was purchased from Pall; 50×TAE, DEPC water, and 30% polyacrylamide were purchased from Sobalife; TB Green™ Premix Ex Taq™ II (TliRNaseH Plus), LA Taq DNA polymerase, and nucleic acid Marker were purchased from Bao Bioengineering Dalian Co., Ltd.; Tween20 and agarose were purchased from Roche. Commercial antibodies used in the present invention include: HRP-labeled goat anti-rabbit IgG antibody (Proteintech), HRP-labeled goat anti-mouse IgG antibody (Proteintech), mouse anti-TPL2 monoclonal antibody (Santa Cruz Biotechnology), mouse anti-β-muscle Actin monoclonal antibody (Santa Cruz Biotechnology); goat anti-rabbit fluorescent secondary antibody (CST). Rabbit anti-SVA polyclonal antibody was provided by the foot-and-mouth disease epidemiology team of Lanzhou Veterinary Research Institute. Western blotting showed three protein bands of VP0, VP1 and VP3.

Instruments: Nucleic acid electrophoresis tank, membrane transfer apparatus and high-resolution image acquisition system were purchased from BIO-RAD Company; CO ₂ constant temperature incubator, 4 °C display cabinet, -20 °C refrigerator, -80 °C ultra-low temperature refrigerator and fluorescence quantitative PCR instrument were all purchased. Purchased from ThermoScientific Company; Laser Scanning Confocal Microscope was purchased from Leica Company, Germany; Inverted Optical Microscope was purchased from Nikon Company; Beijing Liuyi Company; PCR instrument, small room temperature centrifuge, low temperature refrigerated ultracentrifuge and pH meter were purchased from Eppendorf Company.

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. Design of sgRNA oligo sequence

According to the human TPL2 gene sequence (Gene ID: 1326, shown as SEQ ID NO: 1) published in the NCBI database, two pairs of specific sgRNA sequences were designed by the online CRISPR design tool (http://crispr.mit.edu/). , targeting the second exon of human TPL2. The designed oligonucleotide sequences are shown in Table 1.

Table 1 Human TPL2 sgRNA oligo sequences

primer name Primer sequences (5' - 3') serial number H-TPL2-sgRNA1Forward TCCTCGGGGCGCCTTTGGAA SEQ ID NO: 2 H-TPL2-sgRNA1 Reverse TTCCAAAGGCCGCCCCGAGGA SEQ ID NO: 3 H-TPL2-sgRNA2Forward CCGATGTTCTCCTGATCCCC SEQ ID NO: 4 H-TPL2-sgRNA2 Reverse GGGGATCAGGAGAACATCGG SEQ ID NO: 5

2. Construction of pX-EZ-TPL2-sgRNA recombinant plasmid

The synthesized two single-stranded sgRNAoligoDNAs were annealed to form double strands. The CRISPR/Cas9 vector pX459 pSpCas9-2A-puro-MCS and the helper vector EZ-GuideXH were digested with Bbs1 to recover the linearized vector. The annealed sgRNA1 and sgRNA2 double-stranded DNAs were ligated with linearized pX459 pSpCas9-2A-puro-MCS and EZ-GuideXH for 5 min at room temperature (25 °C) with T4 DNA fast ligase to obtain pX459-sgRNA1 and EZ-sgRNA2 recombination. Plasmid, the recombinant plasmid was subsequently sequenced and identified. The results are shown in Figure 1. It can be seen from Figure 1 that the double-stranded DNA of sgRNA1 and sgRNA2 has been successfully connected to pX459 pSpCas9-2A-puro-MCS and EZ-GuideXH. The two recombinant plasmids obtained were double digested with Spe1 and Kpn1. After recovery, the linearized digested products were ligated at room temperature (25 °C) for 5 min with T4 DNA fast ligase, and finally the pX-EZ-TPL2-sgRNA recombinant plasmid was obtained. . The plasmids were transformed into Trans5α competent cells, and then plated on ampicillin-resistant plates at 37°C overnight. The next day, monoclonal bacteria were picked, added with ampicillin-resistant LB medium, and incubated at 37 °C with shaking in an incubator. After 12-16 h, the plasmids were extracted according to the instructions of the plasmid mini-extraction kit for colony PCR screening, and the amplified positive clones were The correct size of the PCR product is about 520bp, which is consistent with the expected size, as shown in Figure 2. Subsequently, the screened positive cloned plasmids were further sequenced and verified with U6 universal primers.

3. Cell culture and transfection

HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and placed in a 37°C, 5% CO ₂ incubator. Before transfection, inoculate HEK293T cells in logarithmic growth phase with good growth status into a 6-well cell culture plate for culture. When the cell density reaches 70% to 80%, 4 μg of the above-constructed pX- The EZ-TPL2-sgRNA recombinant plasmid was transfected into cells (the transfection reagent HighGene and the recombinant plasmid were mixed at a concentration of 2:1), and the same amount of pX-EZ empty vector was transfected as a negative control.

4. Drug screening of monoclonal cell lines

24 to 48 hours after the cells were transfected, the fresh DMEM complete medium containing 1.3 μg/mL puromycin antibiotic was replaced for drug screening, and two days later, the fresh DMEM complete medium containing 0.65 μg/mL puromycin antibiotic was changed to continue screening for about 7 days. All cells in the negative control group died. The obtained positive cells were digested into single cells, and the cells were diluted into 96-well plates by limiting dilution method, and the growth of the single clone was observed after one week of culture, and the single clone with good growth status was selected after about two weeks for identification.

5. Identification of TPL2 knockout HEK293T cell line

The monoclonal cells selected from the 96-well plate were inoculated into a 48-well plate, and when the cells were full, the cells were expanded and cultured to a 24-well plate, a 12-well plate, a 6-well plate and a T25 culture flask in turn. During the period, some cells were taken to extract the total cell RNA and reverse transcribed into cDNA template. High-specific primers designed for the knockout target (see Table 2) were used for PCR amplification and nucleic acid electrophoresis detection. The results are shown in Figure 3. Subsequently, the suspected positive clones were identified by genetyping PCR sequencing and compared with the original genome to detect whether the targeted knockout of the TPL2 gene was successful. The primer sequences for detection are shown in Table 2. The results of sequence comparison analysis showed that the selected HEK293T-KO-TPL2-A2 cell line TPL2 allele 1 was knocked into 54bp between 390 and 391 bp (as shown in SEQ ID NO. 6), and the base at 449 bp was knocked into. "G" was knocked out; the 391-448 bp of allele 2, a total of 58 bp (as shown in SEQ ID NO. 7), was knocked out; HEK293T-KO-TPL2-B1 cell line TPL2 allele 1 of the 393 bp was knocked out The "C" base is knocked out, and the 442-459 bp total 18 bp (as shown in SEQ ID NO. 8) is knocked out, and the 391-448 bp (as shown in SEQ ID NO. 7) of the allele 2 is a total of 58 bp was knocked out, as shown in Figure 4. It can be seen that both HEK293T-KO-TPL2-A2 and HEK293T-KO-TPL2-B1 cell lines are homozygous knockout. The protein was extracted from the correctly sequenced monoclonal cells and control cells, and the expression of TPL2 protein level was detected by Western blotting to further verify the knockout effect of the TPL2 gene. Replication in HEK293T-KO-TPL2 cells". The results showed that the expression of TPL2 protein in the control cell HEK293T-WT-TPL2 was normal, while the expression of TPL2 protein was not detected in the TPL2 knockout monoclonal cell lines HEK293T-KO-TPL2-A2 and HEK293T-KO-TPL2-B1. The expression level of β-actin is normal and basically consistent, and the results are shown in Figure 5, indicating that the present invention successfully established a TPL2 knockout cell line. The HEK293T-KO-TPL2-B1 cell line was randomly selected among the two knockout cell lines for subsequent functional evaluation, and the cell line was named HEK293T-KO-TPL2.

Table 2genetyping PCR detection primer sequences

primer name Primer sequences (5' - 3') H-TPL2 genetyping Forward GACCAGGCACCTGCATCTGTT H-TPL2 genetyping Reverse TGAGGCAGTGCACCCTCAGA

6. Cell morphology observation and proliferation rate analysis of TPL2 gene knockout HEK293T cell line

When HEK293T-KO-TPL2 and HEK293T-WT-TPL2 cells were cultured to 80%-90% confluency, 0.25% trypsin was added for digestion, and when the cells became suspended, fresh DMEM complete medium was added to terminate the digestion. The cell suspension was centrifuged at 1200 r/min for 5 min, and the supernatant was discarded to retain the precipitate. The medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:4. The cells were seeded in 6-well plates, and HEK293T-KO-TPL2 cells grown to monolayer were randomly selected for observation and photographed with an inverted microscope, and their cell morphology was compared with HEK293T-WT-TPL2 cells. TPL2 and HEK293T-WT-TPL2 cells have the same morphology, both adherent and epithelioid cell morphology, as shown in Figure 6. Therefore, knockdown of TPL2 did not result in significant changes in cell morphology under the present experimental conditions. HEK293T-KO-TPL2 and HEK293T-WT-TPL2 cells were continuously passaged in 6-well plates, and the time for the formation of a cell monolayer was observed every 2 passages, and 3 replicates were performed to measure the proliferation rate of cells. Statistical Analysis. The results showed that the second, fourth, sixth, eighth, and tenth passages of HEK293T-KO-TPL2 and HEK293T-WT-TPL2 cell lines had almost no difference in the time to form a cell monolayer, which was about 36 hours. shown in Figure 7. This indicates that both cells have the same proliferation rate.

7. Virus infection

The HEK293T-KO-TPL2 and HEK293T-WT-TPL2 cells were passaged for 2 to 3 generations after self-recovery. After the cells were stable, they were digested and seeded into 6-well plates, with 5×10 ⁵ cells per well, and placed at 37°C, in a 5% CO ₂ incubator. When the cells grew to 80% to 90%, the cells were washed with serum-free DMEM to remove the residual serum in the cells, and then two groups of cells were infected with a multiple multiplicity of infection (MOI) of SVA virus, and placed in the cells. Adsorb for 1 h in a 37 °C, 5% _CO2 incubator. After the adsorption, the inoculum was discarded, replaced with DMEM maintenance medium containing 2% FBS to continue the culture and the cells were harvested at the designated time point. The harvested cell samples were washed twice with PBS to remove adhering virus for subsequent experiments.

8. Indirect immunofluorescence observation of the fluorescence expression of SVA virus particles on HEK293T-KO-TPL2 cells

HEK293T-KO-TPL2 and HEK293T-WT-TPL2 cells were spread in 20 mm glass dishes, and when the cells grew to 60% to 70%, the two groups of cells were infected with SVA virus according to the above infection method and dose; Cells were harvested at 0 h, 8 h, and 16 h, washed three times with 1×PBS; 4% paraformaldehyde (1 mL per dish) was added to fix overnight in the dark; the supernatant was discarded and washed three times with 1×PBS for 5 min each time (lightly add 1×PBS to prevent cells from being washed up); add 0.2% Triton X-100 (0.2% Triton X-100: 100 mL PBS+200 μL Triton X-100) for 1 h at room temperature; discard the supernatant , washed 3 times with 1×PBS, 5 min/time; blocked with 5% BSA (5% BSA: 10 mL + 0.5 g BSA) for 1.5 h at 37 °C; discarded the blocking solution and added SVA virus diluted with 5% BSA The primary antibody was incubated overnight at 4°C; washed three times with 1×PBST (1×PBST: 100 mL 1×PBS+50 μL Tween20) for 10 min/time, and added fluorescein-labeled secondary antibody (immunofluorescence) diluted with 1×PBST. Rabbit secondary antibody), incubate at 37 °C for 1.5 h; wash three times with 1×PBST, 10 min/time, add 100 μL of anti-fluorescence decay mounting medium (containing DAPI) to each dish; observe the fluorescence using a laser confocal instrument, and save Picture. The results are shown in the figure, in the samples collected at 8 h and 16 h after infection, the fluorescence expression of SVA virions in HEK293T-KO-TPL2 cells was significantly higher than that in HEK293T-WT-TPL2 cells, as shown in Figure 8.

9. RT-qPCR to verify the replication of SVA in HEK293T-KO-TPL2 cells

Cells were harvested at 0 h, 8 h, and 16 h after SVA infection, and total cell RNA was extracted by Triozl lysis method, and then the absolute quantitative primers SVA-3D-F/R for amplifying the conserved region of SVA3D protein, and the specific probe of SVA3D protein were used. Absolute quantitative analysis was performed on the extracted whole genome RNA, and the copy number of SVA was determined. The primer sequences are shown in Table 3. The results showed that the copy number of SVA virus SVA3D in HEK293T-KO-TPL2 cells was significantly higher than that in HEK293T-WT-TPL2 cells in samples collected at 8 h and 16 h after infection, as shown in Figure 9. The extracted part of total RNA was reverse transcribed into cDNA, using cDNA as template, using relative quantitative primer SVA-F/R for amplifying SVA, using Mx3005P-QPCR system and TB Green™ Premix Ex Taq™ II (TliRNaseH Plus) reagent Relative quantification of transcript levels of SVA was performed. The expression level of GAPDH mRNA was used as an internal reference value, and the expression level of SVA mRNA was calculated by the 2- ^△△CT method. See Table 3 for the primer sequences. As shown in the figure, in the samples collected at 8 h and 16 h after infection, compared with HEK293T-WT-TPL2 cells, the expression of SVA mRNA in HEK293T-KO-TPL2 cells was significantly increased, as shown in Figure 10.

Table 3 RT-qPCR primer sequences

primer name Primer sequences (5' - 3') SVA-3D Forward AGAATTTGGAAGCCATGCTCT SVA-3DReverse GAGCCAACATAGARACAGATTGC SVA3D probe TTCAAACCAGGAACACTACTCGAGA SVA Forward AGAATTTGGAAGCCATGCTCT SVA Reverse GAGCCAACATAGARACAGATTGC H-GAPDH Forward GACAAGCTTCCCGTTCTCAG H-GAPDH Reverse GAGTCAACGGATTTGGTGGT

10. Western blotting to verify the replication of SVA in HEK293T-KO-TPL2 cells

Cells were harvested at 0 h, 8 h, and 16 h after SVA infection, RAPI cell lysate containing PMSF and 5× protein loading buffer were added, and the cells were scraped off and placed in a metal bath at 100 °C and boiled for 12 min to denature the proteins. Cell debris was then removed by centrifugation at 10,000 r/min for 10 min. The target protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (NC membrane); NC membrane was blocked with 5% nonfat milk powder solution at room temperature for 1-1.5 h; TPL2 antibody ( 1:1000 dilution), SVA antibody (1:1000 dilution) and β-actin antibody (1:5000 dilution), incubate overnight at 4°C on a shaker; wash the membrane 4 times with 1×TBST (8 min/time); add HRP Incubate the labeled goat anti-rabbit IgG (1:5000 dilution) and goat anti-mouse IgG (1:5000 dilution) antibodies at room temperature for 1.5 h; wash the membrane 4 times with 1×TBST (8 min/time); use fully automatic chemiluminescence The imaging analysis system takes pictures and analyzes the electrophoresis results. The results are shown in Figure 11. In the samples collected at 8 h and 16 h after exposure, the abundance of SVA proteins VP0, VP1 and VP3 in HEK293T-KO-TPL2 cells was significantly higher than that in HEK293T-WT-TPL2 cells. These results suggest that knockdown of TPL2 can promote SVA virus replication in HEK293T cells. This is because TPL2 protein has antiviral effect, HEK293T-KO-TPL2 cannot express TPL2 protein correctly, so it is beneficial to the replication of SVA virus.

11. Determination of SVA virus infectivity (TCID ₅₀ )

HEK293T-KO-TPL2 and HEK293T-WT-TPL2 cells were infected with SVA at MOI=1, respectively, and the virus venom was collected when the cytopathic variable reached 50% to 60%. After repeated freezing and thawing in a -80 °C refrigerator for three times, the medium was aspirated and added to a 15 mL centrifuge tube, centrifuged at 5000 r/min for 5 min, and the supernatant was aspirated to use wild-type HEK293T cells for viral infectivity assay. The obtained two groups of SVA viruses were serially diluted by ^10-3 to ^10-10 times with serum-free DMEM, and HEK293T cells that were covered with monolayer in 96-well cell culture plates were inoculated with each dilution of venom, and each dilution was inoculated. 8 wells, 0.1 mL per well. Placed in a 37 °C, 5% CO ₂ constant temperature incubator and observed for 4 days. The cytopathic condition (CPE) was observed and recorded every half day. According to the cytopathic condition of each well, the TCID ₅₀ of the amplified virus was calculated according to the Reed-Muench method. . The results showed that the _{TCID 50} titer of the virus in the supernatant of SVA after replication in HEK293T-WT- ^TPL2 cells was 10 ^5.7 TCID ₅₀ . The determination result was 10 ^6.9 TCID ₅₀ . 0.1 mL ^-1 , as shown in FIG. 12 , an increase of about 16 times. It can be seen that knocking out TPL2 can promote the proliferation of progeny virus after SVA infection, and using HEK293T-KO-TPL2 cells under the same conditions can obtain a higher titer of SVA and increase the production of SVA virus.

The above embodiments are only preferred embodiments of the present invention and do not limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structure, features and principles of the patented scope of the present invention shall be included in the present invention. Invention application patent scope.

SEQUENCE LISTING

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

<120> TPL2 gene knockout HEK293T cell line and its construction method and application

<130> None

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 1404

<212> DNA

<213> People (Homo sapiens)

<400> 1

atggagtaca tgagcactgg aagtgacaat aaagaagaga ttgatttatt aattaaacat 60

ttaaatgtgt ctgatgtaat agacattatg gaaaatcttt atgcaagtga agagccagca 120

gtttatgaac ccagtctaat gaccatgtgt caagacagta atcaaaacga tgagcgttct 180

aagtctctgc tgcttagtgg ccaagaggta ccatggttgt catcagtcag atatggaact 240

gtggaggatt tgcttgcttt tgcaaaccat atatccaaca ctgcaaagca ttttttatgga 300

caacgaccac aggaatctgg aattttatta aacatggtca tcactcccca aaatggacgt 360

taccaaatag attccgatgt tctcctgatc ccctggaagc tgacttacag gaatattggt 420

tctgatttta ttcctcgggg cgcctttgga aaggtatact tggcacaaga tataaagacg 480

aagaaaagaa tggcgtgtaa actgatccca gtagatcaat ttaagccatc tgatgtggaa 540

atccaggctt gcttccggca cgagaacatc gcagagctgt atggcgcagt cctgtggggt 600

gaaactgtcc atctctttat ggaagcaggc gagggagggt ctgttctgga gaaactggag 660

agctgtggac caatgagaga atttgaaatt atttgggtga caaagcatgt tctcaaggga 720

cttgattttc tacactcaaa gaaagtgatc catcatgata ttaaacctag caacattgtt 780

ttcatgtcca caaaagctgt tttggtggat tttggcctaa gtgttcaaat gaccgaagat 840

gtctattttc ctaaggacct ccgaggaaca gagatttaca tgagcccaga ggtcatcctg 900

tgcaggggcc attcaaccaa agcagacatc tacagcctgg gggccacgct catccacatg 960

cagacgggca ccccaccctg ggtgaagcgc taccctcgct cagcctatcc ctcctacctg 1020

tacataatcc acaagcaagc acctccactg gaagacattg cagatgactg cagtccaggg 1080

atgagagagc tgatagaagc ttccctggag agaaacccca atcaccgccc aagagccgca 1140

gacctactaa aacatgaggc cctgaacccg cccagagagg atcagccacg ctgtcagagt 1200

ctggactctg ccctcttgga gcgcaagagg ctgctgagta ggaaggagct ggaacttcct 1260

gagaacattg ctgattcttc gtgcacagga agcaccgagg aatctgagat gctcaagagg 1320

caacgctctc tctacatcga cctcggcgct ctggctggct acttcaatct tgttcgggga 1380

ccaccaacgc ttgaatatgg ctga 1404

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 2

tcctcggggc gcctttggaa 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 3

ttccaaaggc gccccgagga 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 4

ccgatgttct cctgatcccc 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 5

ggggatcagg agaacatcgg 20

<210> 6

<211> 54

<212> DNA

<213> Artificial Sequence

<400> 6

tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaa 54

<210> 7

<211> 58

<212> DNA

<213> Artificial Sequence

<400> 7

ccctggaagc tgacttacag gaatattggt tctgatttta ttcctcgggg cgcctttg 58

<210> 8

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 8

gcctttggaa aggtatac 18

Claims (10)

1. The construction method of TPL2 gene knockout HEK293T cell line is characterized in that, the second exon region of human TPL2 gene is used as the target sequence, specifically, the TPL2 alleles 1 and 1 shown in SEQ ID NO: 1 are used. The 390-459 bp sequence or the 391-448 bp sequence of the second exon of allele 2 was used as the target sequence. 2 . The method for constructing a TPL2 gene knockout HEK293T cell line according to claim 1 , wherein the target sequences of allele 1 and allele 2 are inserted into foreign sequences or knocked out target sequences. 3 . 3. The method for constructing a TPL2 knockout HEK293T cell line according to claim 1, wherein an exogenous sequence is inserted between the 390th to 391bp of the TPL2 allele 1 shown in SEQID NO:1, and Knock out the base "G" at 449 bp, and knock out the 391-448 bp of allele 2, a total of 58 bp; Alternatively, the "C" base of the 393 bp of the TPL2 allele 1 shown in SEQ ID NO: 1 is knocked out, and the 442-459 bp is knocked out for a total of 18 bp, and the 391-448 bp for the knock-out allele 2 is a total of 58 bp. 4. the construction method of TPL2 gene knockout HEK293T cell line according to claim 1, is characterized in that, comprises the following steps: Step 1: Construct two pairs of sgRNAs targeting human TPL2 genome sequence and clone them into the same CRISPR/Cas9 vector plasmid pX459 to obtain pX-EZ-TPL2-sgRNA recombinant plasmid; the above two pairs of sgRNA targeting human TPL2 genome sequence are sgRNA1 and sgRNA2, the sequences of the sgRNA1 and sgRNA2 are synthesized from the sequences shown in SEQ ID NO: 2-3 and SEQ ID NO: 4-5 respectively; Step 2: Transfect the constructed pX-EZ-TPL2-sgRNA recombinant plasmid into HEK293T cells, and kill the negative cells through drug screening; Step 3: Single clone sorting by limiting dilution method; Step 4: The sorted monoclonal cells are expanded and verified. 5. A cell line constructed by the method for constructing a TPL2 gene knockout HEK293T cell line according to any one of claims 1 to 4. 6. The cell line according to claim 5, wherein the cell line is the cell line HEK293T-KO-TPL2 whose deposit number is CCTCC NO: C2019328. 7. The application of the cell line according to claim 6 in the study of the way that TPL2 inhibits viral replication and the pathogenic mechanism of the virus. 8. The application of the cell line according to claim 6 in the study of the mode of TPL2 inhibiting virus replication and the pathogenic mechanism of the virus, wherein the virus is SVA. 9. The application of the cell line of claim 6 in separating and culturing SVA. 10. The application of the TPL2 gene knockout HEK293T cell line of claim 6 in the large-scale cellular culture and production of SVA vaccine strains.

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