CN112877275A

CN112877275A - HDAC2 gene knockout BHK-21 cell line and construction method and application thereof

Info

Publication number: CN112877275A
Application number: CN202110154125.XA
Authority: CN
Inventors: 孙跃峰; 龚青; 侯石桐; 殷相平; 王相伟; 毛箬青
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2021-02-04
Filing date: 2021-02-04
Publication date: 2021-06-01
Anticipated expiration: 2041-02-04
Also published as: CN112877275B

Abstract

The present invention discloses a method for constructing a BHK-21 cell line with HDAC2 gene knockout, using CRISPR/Cas9 technology to knock out HDAC2 in the cell line BHK-21 for foot-and-mouth disease vaccine production; After the CRISPR plasmid was transfected into BHK‑21 cells, multiple cell clones were isolated by the cloning loop method using antibiotic screening combined with gradient dilution. After extraction of genomic DNA, PCR amplification and sequencing were performed, and a homozygous transfer of HDAC2 gene was successfully identified. A cell clone with a mutation in the code; wherein HDAC2‑KO‑B3 has a 1-base deletion at the predetermined cleavage position of Cas9. In the HDAC2-knockout BHK-21 cell line, the replication rate of foot-and-mouth virus was significantly accelerated, and the final virus titer was significantly increased, and HDAC2-knockout had no significant effect on the cell growth rate, indicating that it has the prospect of being used for the production of foot-and-mouth disease vaccine. This lays the foundation for further knocking out HDAC2 in suspension cultured BHK-21 cells for direct use in the production of foot-and-mouth disease vaccines.

Description

HDAC2 gene knockout BHK-21 cell line and construction method and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a BHK-21 cell line with an HDAC2 gene knocked out, a construction method and application thereof.

Background

Foot-and-mouth disease (FMD) is an acute, hot and highly contagious epidemic disease caused by the infection of FMDV (Foot-and-mouth disease virus), and mainly infects cloven-hoofed animals such as pigs, cows, sheep and the like. The disease has multiple transmission ways and high transmission speed, once causes huge economic loss in the world, and is listed as the first epidemic disease of A-type animals by the world animal health organization. At present, the prevention and control of the disease are mainly based on vaccine immunization prevention, and the traditional inactivated vaccine still occupies the market leading position. The production of inactivated foot-and-mouth disease vaccine is completely dependent on the replication of foot-and-mouth disease virus in Baby hamster kidney passaged cell BHK-21(Baby hamster kidney cell). BHK-21 cells were first established by MacPherson and Stoker in 1962 using 1 day old Syrian hamster kidney cells. The cell grows rapidly, the virus sensitivity spectrum is wide, and the cell is subsequently used for the proliferation of various viruses and the production of vaccines, such as foot-and-mouth disease vaccines, rabies vaccines, newcastle disease vaccines and the like. The original BHK-21 cells are anchorage-growing cells, which are not favorable for large-scale production of commercial vaccines. The Capstic et al domesticated BHK-21 cells in suspension culture, and cultured the domesticated suspension culture type BHK-21 cells in a stainless steel fermentation tank in 1965 to produce the foot and mouth disease vaccine, and opened a new era of producing the foot and mouth disease vaccine by using the suspension culture BHK-21 cells. The production process of foot-and-mouth disease vaccine in China is that BHK-21 cells cultured by adherence are inoculated with foot-and-mouth disease virus for production after multiple subculture and expansion. The foot-and-mouth disease vaccine production enterprises in China started to perform the technological transformation of BHK-21 cell suspension culture in sequence in 2009, and the bioreactor suspension culture technology is realized in short several years. At present, the adherent growth type BHK-21 cells are mainly used for early-stage research in a laboratory, and the suspension culture type BHK-21 cells are completely adopted for commercial vaccine production. Although BHK-21 cells have been used for more than 50 years in the production of foot and mouth disease vaccines, except suspension culture and domestication, no research has been made to modify BHK-21 cells by genetic means so that the BHK-21 cells are more favorable for virus replication and the BHK-21 cells after genetic modification are used in the production of foot and mouth disease vaccines.

The CRISPR/Cas9 technology is a novel gene editing technology which is rapidly developed in recent years and can be used for mammalian cells, and has a very good application prospect in many fields. The technology is used for modifying related genes for regulating and controlling foot-and-mouth disease virus infection and immunity in BHK-21 cells, so that the replication efficiency of the foot-and-mouth disease virus in the BHK-21 cells is improved, and the yield or the quality of the foot-and-mouth disease vaccine can be improved.

Protein acetylation is an important protein posttranslational modification, and existing researches show that the protein acetylation influences the infection and immune processes of viruses in various ways such as acetylation of virus self proteins, acetylation of host immune related gene promoter region histones, acetylation of immune signal molecules and the like. The level of protein acetylation is dynamically regulated by Histone Acetyltransferase (HAT) and Histone Deacetylase (HDAC). The HDACs family in mammals shares 18 members, and different HDACs have specific functions, with no apparent functional redundancy. Individual members of the HDACs family have been shown to play important regulatory roles in the virus-host interaction process. However, the role of protein acetylation and HDACs family genes in the foot and mouth disease virus infection process is unclear.

Disclosure of Invention

The invention aims to provide a gene-edited BHK-21 cell line which is expected to be used for producing a foot-and-mouth disease vaccine. By using the CRISPR/Cas9 technology, after the HDAC2 is knocked out from the BHK-21 cells, the replication rate of the foot-and-mouth disease virus is accelerated, the virus titer is obviously improved, and the method is expected to be used for producing the foot-and-mouth disease vaccine and improving the yield and the quality of the vaccine.

The foot-and-mouth disease virus used in the invention is an epidemic strain FMDV/O/BY/2010 in China in recent years, and the strain is also a vaccine strain at present. The cell line used in the invention is an adherent culture type BHK-21 cell line. Cell culture in 5% CO₂In the incubator, the temperature is 37 ℃, and 10% fetal bovine serum and 1% antibiotic (penicilin-streptomycin) are added into a DMEM medium. The specific technical scheme is as follows:

1. construction of HDAC2 knockout BHK-21 cell line by using CRIPSR/Cas9 technology

Based on the HDAC2 gene sequence of the golden hamster (Mesocricetus auratus) in NCBI, gRNA sequences were designed in the 1 st exon region of HDAC2 using CRISPOR software (http:// CRISPOR. for. net /): ATGGCGTACAGTCAAGGAGG are provided. The designed gRNA was synthesized, annealed and ligated to PX459(addge #62988) plasmid according to methods published by the zhanfeng laboratories (Nature Protocols, 2013). And extracting the CRISPR plasmid with correct sequencing for later use. According to the standard transfection procedure of Invitrogen Lipofectamine 2000, the CRISPR plasmid is used for transfecting a BHK-21 cell line, puromycin with the final concentration of 3 mu g/mL is added after 48 hours of transfection, after 5-7 days of screening, the cells are counted, 100 cells and 300 cells are respectively paved, after one week of single cell clone formation, the single cell is picked by using a cloning ring, transferred to a 24-well plate and subjected to amplification culture. Different cell clones were collected and characterized at the DNA level and at the protein level, respectively. When the DNA level is identified, the following primers are used for amplification: GT-FP (AGTACACCCAAACCTGTGTGGC), GT-RP (CTACACTCCTTCAGCCCCAC), and Sanger sequencing of the amplification products. Protein level identification the protein expression level of HDAC2 was measured using Western blot using antibodies to HDAC2 (CST, 57156).

2. Foot-and-mouth disease virus infection experiment and virus replication rate evaluation

HDAC2 knock-out BHK-21 and control cell lines (BHK-21 cell lines transfected with PX459 empty plasmid) were cultured in a 60mm cell culture dish to reach approximately 80% confluence, the medium was aspirated, 2mL PBS was added to wash the cells twice and then the PBS was aspirated and cleaned, 1mL dmdmdmdmmem (control) or foot and mouth disease virus diluted to MOI 0.1 was added, incubated in an incubator for 1h, the virus solution was aspirated, 2mL PBS was added to wash the cells twice and then the PBS was aspirated and cleaned, and 3mL DMEM medium was added. And respectively collecting supernatant and cell samples at different infection times, and comprehensively evaluating the replication condition of the foot-and-mouth disease virus by using methods such as RT-qPCR, Western blot, virus titer determination and the like.

RT-qPCR detection of relative expression of viral RNA:

the collected cell samples were subjected to Trizol method to extract total RNA, and after concentration measurement, PrimeScript was used^TMRT reagent Kit with gDNA Eraser (TaKaRa, RR047A) reverse transcription Kit reverse transcription, qPCR was done with SYBR Green qPCR Supermix (Takara) reagent. beta-Actin was used as an internal reference gene to quantify the relative expression level of VP 1. The primers used were: VP1-q-FP (GACAACACCACAACCCA), VP1-q-RP (CCTTCTGTAGCCAGCAGCACTT); beta-actin-q-FP (GCTGGCCGGGACCTGACAGATCCC), beta-actin-q-RP (TCTCCAGGGAGGAAGGATGCGGCGGC).

Westernblot for detecting the protein expression level of the foot-and-mouth disease virus structural protein VP 1:

170 μ l of cell lysate (Pierce) was added to the collected cell sample, the cell debris was removed by centrifugation after the cells were sufficiently lysed to collect the supernatant, and total protein was quantified using BCA protein quantification kit (Thermo). The remaining portion was mixed with a quarter volume of 4 Xloading buffer, boiled for 5 minutes, and allowed to stand at room temperature for cooling. The quantitative results were used to determine the loading volume, which was typically 20-40. mu.g. Protein samples are subjected to SDS-PAGE electrophoresis, then transferred to a PVDF membrane, and transferred to the PVDF membrane at a constant pressure of 90V in ice bath for 2 h. Sealing with 5% skimmed milk powder for 1 hr. beta-Actin antibody (Santa Cruz) was diluted 1:6000 times, antibody to VP1 (supplied by the institute Zheng Hai laboratory) was diluted 1:1000 times, and primary antibody was incubated overnight at 4 ℃. Horseradish peroxidase (HRP) -labeled corresponding secondary antibody was diluted 1:4000 fold and incubated for 1h at room temperature. Protein detection was performed using a chromogenic kit (Pierce) from Thermo corporation^TMECLWestern Blotting Substrate), and the film was placed in a film imaging system to collect corresponding protein pictures.

Determination of viral titres:

BHK-21 cells were grown in 96-well microplates to a confluence of about 70% and ready for use. Firstly, virus liquid to be detected is respectively diluted by 10 times in a gradient way in a centrifugal tube of 1.5mL, cells are washed twice by 100 mu l PBS, and the diluted virus liquid is used forVirus fluid was inoculated into 96-well plates in a column of 8 wells per dilution, and 100 μ l was inoculated per well. Incubate for 1h in the incubator, aspirate the virus, wash the cells twice with 100 μ l PBS, blot clean PBS, add maintenance medium containing 1% FBS, and continue incubation in the incubator. Observing and recording cytopathic results day by day, and calculating TCID according to Reed-Muench two-handed method after continuously observing for 5-7 days₅₀The value is obtained.

3. Determination of cell growth rate

After digestion, the cells are counted, 10000 cells per well are inoculated to a 96-well plate, and the plate is placed in a cell culture box for normal culture. To determine cell viability at a particular time, 10. mu.L of CCK8 solution was added to each well of medium, the incubator was placed for further incubation for 1h, and the absorbance at 450nm was measured on a Varioskan LUX multifunctional microplate reader (Thermo). A growth curve of the cells was prepared based on the measured absorbance.

The applicant takes the frequently used adherent growth type BHK-21 cells in a laboratory as an object, and discovers that HDAC2 has a very obvious effect of resisting foot and mouth disease virus infection by performing early stage screening on the genes of the HDACs family and constructing an HDAC gene knockout cell line by using the CRISPR/Cas9 technology. The replication rate of the hoof virus in the BHK-21 cell line knocked out by HDAC2 is obviously accelerated, the final virus titer is obviously improved, and the cell growth rate is not obviously influenced after the HDAC2 is knocked out, which indicates that the cell has a prospect for producing foot-and-mouth disease vaccines. Lays a foundation for further knocking out HDAC2 in suspension culture type BHK-21 cells and directly applying the cells to the production of the foot-and-mouth disease vaccine.

Has the advantages that:

the invention successfully constructs the BHK-21 cell line knocked out by HDAC2 gene by using CRISPR/Cas9 technology; the HDAC2 gene is proved to have very obvious effect of resisting hoof and mouth disease virus infection for the first time, the replication rate of the foot and mouth disease virus in the BHK-21 cell line knocked out by the HDAC2 is accelerated, the virus titer is obviously improved, and the cell growth rate is not obviously influenced, so that the HDAC2 gene is expected to be used for the commercial production of the foot and mouth disease vaccine.

Drawings

Figure 1 shows the identification of HDAC2 knock-out cell line.

FIG. 1A shows the partial sequence alignment of wild-type HDAC2(WT) in BHK-21 cells and mutant HDAC2 near the mutation position in a knock-out cell line.

FIG. 1B shows the sequencing peaks near the mutation position of wild-type HDAC2 in BHK-21 cells and mutant HDAC2 in knock-out cell lines.

Fig. 1C demonstrates success at the protein level for HDAC2 gene knockout. Collecting normal culture cells of a control cell line and an HDAC2 knockout cell line, detecting the expression level of endogenous HDAC2 protein by Western blot, and taking beta-Actin as an internal reference control.

Figure 2 shows that the rate of replication of foot and mouth disease virus was significantly accelerated in HDAC2 knock-out cell line.

Figure 2A shows that the level of viral RNA replication in the HDAC2 knock-out cell line (HDAC2-KO-B3) was significantly higher than the control cell line (WT) following foot and mouth disease virus infection. Different cell lines are infected by foot-and-mouth disease virus with 0.1MOI for different time, cell samples are collected, and virus VP1 RNA is relatively quantified by using an RT-qPCR method.

FIG. 2B shows that protein accumulation of VP1 is significantly accelerated in HDAC2 knock-out cell line (HDAC2-KO-B3) after foot and mouth disease virus infection. Different cell lines were infected with foot-and-mouth disease virus at 0.1MOI, and cell samples were collected at different times, and the protein level of VP1 was measured using Western blot, and β -Actin was used as an internal control.

Figure 2C shows that viral titers were significantly elevated in HDAC2 knockout cell line medium following foot and mouth disease virus infection. Infecting different cell lines with 0.1MOI of foot and mouth disease virus, culturing for different time, collecting cell culture medium, and treating with TCID₅₀The method determines the virus titer.

Figure 3 shows that the growth rate of cells was not significantly affected following HDAC2 knockdown. The control cell line and the HDAC2 knockout cell line were inoculated in a 96-well plate and cultured normally, and cell viability was measured at regular intervals by the CCK8 method to prepare a growth curve.

Detailed Description

The invention is further described below with reference to the accompanying drawings:

HDAC2 gene knockout BHK-21 cell line successfully constructed by CRISPR/Cas9 technology

According to preliminary screening and preliminary functional analysis of HDAC family members in our laboratory, the inventors found that HDAC2 has an important effect of resisting foot-and-mouth disease virus infection. Therefore, the inventor intends to perform gene knockout on HDAC2 in a cell line BHK-21 for producing the foot-and-mouth disease vaccine by using the CRISPR/Cas9 technology so as to improve the replication efficiency of the foot-and-mouth disease virus in the cell line and improve the yield or quality of the foot-and-mouth disease vaccine. After transfecting BHK-21 cells with CRISPR plasmid for knocking out HDAC2, the inventor separates a plurality of cell clones by a cloning loop method by using antibiotic screening and gradient dilution. After extracting genomic DNA, PCR amplification and sequencing are carried out, and 1 cell clone with homozygous frameshift mutation of HDAC2 gene is successfully identified. Wherein HDAC2-KO-B3 had a deletion of 1 base at the Cas9 predetermined cleavage position (fig. 1A). Such frameshift mutations are expected to cause frameshift, premature termination and loss of function in protein translation. The quality of the sequencing peak image near the mutation position is high (FIG. 1B), which indicates that the sequencing result is reliable. To further confirm the gene knockout of HDAC2, the inventors collected normal culture cell samples of the control cell line and HDAC2 knockout cell line, and detected the expression level of endogenous HDAC2 protein using Western blot, as shown in fig. 1C, the expression of HDAC2 could be detected in the control cell line, and HDAC2 could not be detected in the knockout cell line, which proves that the inventors succeeded in obtaining a BHK-21 cell line with HDAC2 gene completely knocked out.

Example 1

Test example 1

The replication rate of the foot-and-mouth disease virus in the HDAC2 knockout cell line is obviously accelerated

In order to evaluate the replication rate of the foot-and-mouth disease virus in the HDAC2 knockout cell line, the inventor uses the foot-and-mouth disease virus with 0.1MOI to respectively infect a control cell line and an HDAC2 knockout cell line, cultures samples are collected at different times, respectively detects the replication level of virus RNA, the accumulation of virus protein and the virus titer, and comprehensively evaluates the influence on the replication rate of the foot-and-mouth disease virus after the HDAC2 knockout. As shown in fig. 2A, following foot and mouth disease virus infection, the level of viral RNA replication in the HDAC2 knock-out cell line (HDAC2-KO-B3) was significantly higher than the control cell line (WT). As shown in FIG. 2B, when the cells were infected with foot-and-mouth disease virus, the protein level of VP1 was detected by Westernblot, and β -Actin was used as an internal reference control, it was determined that the expression level of virus structural protein VP1 was significantly increased in the HDAC2 knockout cell line (HDAC2-KO-B3) compared to the control cell line (WT). The virus titer results showed that the virus titer of HDAC2 knockout cell line was significantly increased after foot and mouth disease virus infection compared to the control cell (fig. 2C). These results fully demonstrate that HDAC2 knockout cells can significantly accelerate the rate of replication of foot and mouth disease virus.

Test example 2

There was no significant difference in growth rate between the HDAC2 knockout cell line and the control cell line

HDAC2 knock-out cells (HDAC2-KO-B3) and a control cell line (WT) were seeded in a 96-well plate for normal culture at 10000 cells per well by cell counting. After 6h, 12h, 24h and 36h of plating, 10ul of CCK8 solution is added into each well, cultured for 1h, and the absorbance value of 450nm is measured. And taking the time and the OD value as an abscissa and an ordinate to make a growth curve. From the results, there was no significant difference in growth rate of the HDAC2 knock-out cell line compared to the control group (fig. 3).

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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Claims

A method for constructing a HDAC2 gene knock-out BHK-21 cell line, comprising: based on the HDAC2 gene sequence of CRISPOR in cricket in NCBI, gRNA sequences were designed in the 1 st exon region of HDAC2 using CRISPOR software: ATGGCGTACAGTCAAGGAGG, respectively; the designed gRNA was synthesized, annealed and ligated to PX459(Addgene #62988) plasmid; extracting CRISPR plasmid with correct sequencing for later use; according to a standard transfection procedure of Invitrogen Lipofectamine 2000, a BHK-21 cell line is transfected by CRISPR plasmid, puromycin with the final concentration of 3 mug/mL is added after 48 hours of transfection, the cell is counted after 5-7 days of screening, 100 cells and 300 cells are respectively paved, after one week, a single cell is cloned and then a single clone is picked up by using a cloning ring, transferred to a 24-well plate and subjected to expanded culture; different cell clones were collected and characterized at the DNA level and at the protein level, respectively.
2. The method of constructing an HDAC2 gene knockout BHK-21 cell line according to claim 1, wherein: the cell line is an adherent culture type BHK-21 cell line; cell culture in 5% CO₂In the incubator, the temperature is 37 ℃, and 10% fetal bovine serum and 1% antibiotic (penicilin-streptomycin) are added into a DMEM medium.
3. The method of constructing an HDAC2 gene knockout BHK-21 cell line according to claim 1, wherein: when the DNA level is identified, the following primers are used for amplification: GT-FP (AGTACACCCAAACCTGTGTGGC), GT-RP (CTACACTCCTTCAGCCCCAC), and Sanger sequencing the amplification product; protein level identification using an antibody to HDAC2 (CST, 57156), the protein expression level of HDAC2 was measured using Westernblot.
4. The method of constructing an HDAC2 gene knockout BHK-21 cell line according to claim 1, wherein: obtaining an HDAC2 knock-out cell HDAC2-KO-B3, HDAC2-KO-B3 having a 1 base deletion at a predetermined cleavage position of Cas 9; the protein expression level of endogenous HDAC2 is detected by Western blot, and the protein expression of HDAC2 cannot be detected by the HDAC2-KO-B3 cell line.
5. An HDAC2 knock-out BHK-21 cell line constructed according to the method of any one of claims 1-4.
6. The method for evaluating the replication rate of foot-and-mouth disease virus in HDAC2 gene-knocked-out BHK-21 cell line of claim 5, wherein:

when an HDAC2 knockout BHK-21 cell line and a control cell line (BHK-21 cell line transfected with PX459 empty plasmid) are cultured in a 60mm cell culture dish to reach about 80% confluence, sucking out the culture medium, adding 2mL of PBS to wash the cells twice, sucking out the PBS completely, adding 1mL of DMEM (control) or foot and mouth disease virus diluted to MOI (MOI) 0.1, incubating for 1h in an incubator, sucking out the virus solution, adding 2mL of PBS to wash the cells twice, sucking out the PBS completely, and adding 3mL of DMEM culture medium; respectively collecting supernatant and cell samples at different infection times, and comprehensively evaluating the replication condition of the foot-and-mouth disease virus by using RT-qPCR, Western blot and virus titer determination methods;

RT-qPCR detection of relative expression of viral RNA:

the collected cell samples were subjected to Trizol method to extract total RNA, and after concentration measurement, PrimeScript was used^TMThe RT reagent Kit with gDNA Eraser (TaKaRa, RR047A) reverse transcription Kit carries out reverse transcription, and qPCR is completed by SYBR Green qPCR Supermix (Takara) reagent; taking beta-Actin as an internal reference gene to quantify the relative expression level of VP 1; the primers used were: VP1-q-FP (GACAACACCACAACCCA), VP1-q-RP (CCTTCTGTAGCCAGCAGCACTT); β -actin-q-FP (GCTGGCCGGGACCTGACAGATACCC), β -actin-q-RP (TCTCCAGGGAGGAAGGATGCGGCGGC);

westernblot for detecting the protein expression level of the foot-and-mouth disease virus structural protein VP 1:

adding 170 mu l of cell lysate (Pierce) into the collected cell sample, centrifuging to remove cell debris after the cells are fully lysed, collecting supernatant, and quantifying total protein by using a BCA protein quantification kit; adding one fourth volume of 4 Xloading buffer solution into the rest part, mixing, boiling for 5 min, standing at room temperature, and cooling; determining the sample loading volume by using the quantitative result, wherein the sample loading amount is 20-40 mu g; protein samples are subjected to SDS-PAGE electrophoresis, then converted into a PVDF membrane, and converted into the PVDF membrane in ice bath at a constant voltage of 90V for 2 hours; sealing with 5% skimmed milk powder for 1 hr; 1:6000 times dilution of beta-Actin antibody (Santa Cruz) and 1:1000 times dilution of antibody of VP1, and incubating overnight at 4 ℃; horseradish peroxidase (HRP) -labeled corresponding secondary antibody is diluted 1:4000 times, and the secondary antibody is at room temperatureIncubating for 1 h; protein detection was performed using a chromogenic kit (Pierce) from Thermo corporation^TMECL Western Blotting Substrate), placing the membrane in a membrane imaging system to collect corresponding protein pictures;

determination of viral titres:

BHK-21 cells were grown in 96-well microplates to a confluence of about 70% for future use; firstly, respectively carrying out continuous 10-time gradient dilution on virus liquid to be detected in a 1.5mL centrifuge tube, washing cells twice by using 100 mu l PBS, inoculating the diluted virus liquid into a 96-well plate, and inoculating a row of virus liquid with 8 holes in each dilution degree and 100 mu l of virus liquid in each hole; incubating for 1h in an incubator, sucking virus liquid, washing cells twice with 100 mu l of PBS, sucking clean PBS, adding a maintenance culture medium containing 1% FBS, and continuously culturing in the incubator; observing and recording cytopathic results day by day, and calculating TCID according to Reed-Muench two-handed method after continuously observing for 5-7 days₅₀The value is obtained.
7. The method for determining the cell growth rate of an HDAC2 gene knock-out BHK-21 cell line of claim 5, wherein:

counting after cell digestion, inoculating 10000 cells per hole to a 96-hole plate, and placing the 96-hole plate in a cell culture box for normal culture; to determine the cell viability at a specific time, 10. mu.L of CCK8 solution was added to each well of the medium, the incubator was placed for further incubation for 1h, and the absorbance at 450nm was measured on a Varioskan LUX multifunctional microplate reader; a growth curve of the cells was prepared based on the measured absorbance.
8. The use of the HDAC2 gene knock-out BHK-21 cell line of claim 5 in the production of a foot and mouth disease vaccine.
9. A method for knocking out HDAC2 gene based on CRISPR/Cas9 technology is characterized in that: the method comprises the following steps: based on the HDAC2 gene sequence of CRISPOR in cricket in NCBI, gRNA sequences were designed in the 1 st exon region of HDAC2 using CRISPOR software: ATGGCGTACAGTCAAGGAGG, respectively; the designed gRNA was synthesized, annealed and ligated to PX459(Addgene #62988) plasmid; extracting CRISPR plasmid with correct sequencing for later use; the CRISPR plasmid transfected cell lines were according to standard transfection procedure of Invitrogen Lipofectamine 2000.
10. The HDAC2 gene knockout cell line obtained by the method for knocking out the HDAC2 gene based on the CRISPR/Cas9 technology is applied to the production of the foot-and-mouth disease vaccine.