CN110577934A

CN110577934A - Construction method and application of TLR4 gene knocked-down porcine alveolar macrophage cell line

Info

Publication number: CN110577934A
Application number: CN201910871910.XA
Authority: CN
Inventors: 郑海学; 李丹; 张敬; 杨文萍; 李露露; 茹毅; �田宏; 张克山; 杨帆; 刘湘涛
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2019-09-16
Filing date: 2019-09-16
Publication date: 2019-12-17
Anticipated expiration: 2039-09-16
Also published as: CN110577934B

Abstract

The invention relates to the technical field of biology, in particular to a construction method of a TLR4 gene knocked-down porcine alveolar macrophage cell line and application thereof. The preservation number of the porcine alveolar macrophage cell line is as follows: CCTCC NO: C2019174. the construction method of the TLR4 gene knocked-down porcine alveolar macrophage cell line comprises the following steps: transfecting porcine alveolar macrophages with recombinant lentiviruses containing sgRNA, and obtaining the TLR4 gene-knocked-down porcine alveolar macrophage system through resistance screening; the sgRNA is obtained by annealing a nucleotide sequence shown in SEQ ID NO. 1-2. The TLR4 knocked-down gene porcine alveolar macrophage cell line constructed by the invention can promote replication of foot-and-mouth disease viruses, and compared with an undeknocked cell line, the replication amount of the foot-and-mouth disease viruses in the TLR4 knocked-down gene porcine alveolar macrophage cell line is 2-3 times that of the same.

Description

Construction method and application of TLR4 gene knocked-down porcine alveolar macrophage cell line

Technical Field

The invention relates to the technical field of biology, in particular to a construction method of a TLR4 gene knocked-down porcine alveolar macrophage cell line and application thereof.

background

Foot and Mouth Disease (FMD) is a highly contagious disease that affects mainly pigs, cattle, sheep, goats, deer and other artiodactyls. The pathogenic agent of foot-and-mouth disease is foot-and-mouth disease virus (FMDV), belonging to the genus of foot-and-mouth disease virus of the family Picornaviridae. The viral genome comprises a positive single-stranded RNA strand 8.5kb in length, encoding four structural proteins (VP1-VP4), eight non-structural proteins (L, 2A, 2B, 2C, 3A, 3B, 3C and 3D) and some cleavage intermediates. Viral proteins have multiple functions, and can achieve the transmission and replication of FMDV through many different mechanisms and counteract the host's antiviral response.

TLRs are type I transmembrane proteins composed of an extracellular leucine-rich repeat domain, an intracellular conserved Toll/IL-1 receptor domain, and a transmembrane domain. TLRs are the first immunological defense line of organisms against external microbial invasion, are also bridges for connecting innate immunity and adaptive immunity, and belong to a pattern recognition receptor family. At present, 13 TLRs have been identified, which are widely distributed and mainly expressed in various immune cells, such as dendritic cells, macrophages, neutrophils, lymphocytes, microglia and the like. TLRs recognize microorganisms including bacteria, viruses, fungi, and the like, and bind to their conserved sequences. Once activation of TLRs is complete, a series of conserved cascades begins and eventually activates two major intracellular transcription factors: NF-. kappa.B and interferon regulatory factors.

toll-like receptor 4(TLR4) belongs to the Pattern Recognition Receptor (PRR) family. They are highly conserved receptors that recognize conserved pathogen-associated molecular patterns (PAMPs) and thus serve as the first line of defense against infection. TLR4 has long been known as a receptor for gram-negative Lipopolysaccharide (LPS). In addition, it binds to endogenous molecules produced by tissue damage. Thus, TLR4 is a key receptor, and both infectious and non-infectious stimuli induce pro-inflammatory responses. TLR 4-mediated inflammation triggered by exogenous or endogenous ligands is also implicated in several acute and chronic diseases, with a key role as an amplifier of the inflammatory response.

The gene of TLR4 is localized to chromosome 9, is mainly distributed in mononuclear macrophages, and can recognize bacterial Lipopolysaccharide (LPS), bacterial teichoic acid, heat-shock proteins (HSPs) released by host necrotic cells, and the like. The TLR4 gene is identified on the surface of human cells for the first time, consists of three parts of extracellular region, transmembrane region and intracellular region, has a total length of 2526bp CDS sequence, comprises three exons and can code 841 amino acids in livestock. Through research in recent years, more biological functions of TLR4 have been discovered, which mainly include inducing immune inflammatory factors to trigger inflammatory response, inducing specific immunity of livestock and poultry bodies, assisting bodies to clear in vivo viruses and generating synergistic effect with other TLR molecules (Molteni Monica, GemmaAbrina, Rosseti Carlo. the Role of Toll-Like Receptor4 in infections and nocinguctions infection. [ J ]. Mediators of infections, 2016,2016.). Most of the functions of the traditional Chinese medicine are involved in the immune response of the organism, so that the traditional Chinese medicine is mainly expressed in cells involved in host defense functions, such as mononuclear macrophages, granulocytes, dendritic cells, lymphocytes, epithelial cells, endothelial cells, bone marrow mononuclear cells and the like in the organism of livestock and poultry.

the CRISPR-Cas9 gene editing technology is a third-generation genome editing technology which is rapidly developed after ZFN and TALEN technologies, is an adaptive immune defense formed by bacteria and archaea in the long-term evolution process, and can be used for resisting invading viruses and exogenous DNA. The CRISPR-Cas9 gene editing technology is a technology for carrying out specific DNA modification on a target gene, and shows great application prospect in the application field of a series of gene therapies.

Disclosure of Invention

The invention obtains the TLR4 gene knocked-down porcine alveolar macrophage cell line by using a CRISPR-Cas9 gene editing technology, and the obtained cell line can be used for promoting the replication of foot-and-mouth disease viruses.

The invention specifically adopts the following technical scheme:

The invention provides a TLR4 gene knocked-down porcine alveolar macrophage cell line, which has a preservation number of CCTCC NO: C2019174.

The invention also provides a construction method of the TLR4 gene-knocked-down porcine alveolar macrophage cell line, which comprises the steps of transfecting the porcine alveolar macrophage cell with recombinant lentivirus containing sgRNA, and obtaining the TLR4 gene-knocked-down cell line through resistance screening; the sgRNA is obtained by annealing a nucleotide sequence shown in SEQ ID NO. 1-2.

Further, the construction method of the TLR4 gene knocked-down porcine alveolar macrophage cell line specifically comprises the following steps:

Step 1: designing a sgRNA primer F and a sgRNA primer R according to a porcine TLR4 gene sequence; the nucleotide sequence of the sgRNA primer F is shown in SEQ ID NO:1, the nucleotide sequence of the sgRNA primer R is shown in SEQ ID NO:2 is shown in the specification;

step 2: enzyme digestion of a lentiviral vector, annealing a sgRNA primer F and a sgRNA primer R to obtain a sgRNA, and connecting the enzyme digestion lentiviral vector with the sgRNA, wherein the lentiviral vector is pGL-U6-gRNA;

and step 3: packaging the recombinant lentiviral vector obtained in the step 2;

And 4, step 4: infecting porcine alveolar macrophages with the lentiviruses obtained in the step 3, and screening to obtain the porcine alveolar macrophage system with the knocked-down TLR4 gene.

Further, the reaction system for annealing the sgRNA primers F and R is as follows: sgRNA primer F1 μ L, sgRNA primer R1 μ L, annealing buffer48 μ L; reaction procedure: 90 deg.C, 4min, 70 deg.C, 10min, 37 deg.C, 20min, 10 deg.C, 20 min.

The invention also provides a lentiviral vector for knocking down TLR4 gene expression, wherein the lentiviral vector comprises the sgRNA.

The TLR4 gene knocked-down porcine alveolar macrophage cell line can be used for promoting the replication of foot-and-mouth disease viruses.

The invention has the beneficial effects that:

1. The sgRNA primer pair for knocking down the TLR4 gene of the PAM cell is constructed, and compared with a control primer pair, the knock-down efficiency of the primer pair is obvious.

2. Compared with an untargeted cell line, the replication capacity of the foot-and-mouth disease virus in the TLR4 gene knocked-down alveolar macrophage cell line prepared by the invention is 2-3 times of that of the porcine alveolar macrophage cell line.

Drawings

Fig. 1 is a graph of the effect of sgRNA knockdown constructed from three pairs of sgRNA primers.

FIG. 2 shows the dose-dependent degradation of VP3 protein by the PRK-HA-TLR4 plasmid.

Fig. 3 shows the 3D gene expression of FMDV after FMDV infection with PAM cells overexpressing TLR 4.

FIG. 4 shows the protein content of VP3 in FMDV-infected cells after infection with PAM cells overexpressing TLR 4.

FIG. 5 shows the results of TCID50 detection after FMDV infection of PAM cells overexpressing TLR 4.

fig. 6 shows the 3D gene expression of FMDV in wild-type PAM cells and PAM cells knocked-down for TLR4 after FMDV infection with PAM cells.

FIG. 7 shows the content of VP3 protein in wild-type PAM cells and PAM cells knocked-down for TLR4 after FMDV infection of PAM cells.

Fig. 8 shows TCID50 assay results of wild-type PAM cells and TLR4 knocked-down PAM cells after FMDV infection with PAM cells.

FIG. 9 is a plasmid map of pCDNA3.1-HA-N.

FIG. 10 is a plasmid map of pCDNA3.1-3 Xflag-C.

In FIGS. 1,2, 4 and 7, α TLR4 represents an antibody against endogenous TLR4, α β -actin represents an antibody against intracellular reference, α Flag represents a Flag-tagged antibody, α HA represents an HA-tagged antibody, TLR4 represents an expression level of endogenous TLR4, β -actin represents an expression level of intracellular reference, Flag-VP3 represents that VP3 is Flag-tagged and VP3 is present, HA-TLR4 represents that TLR4 is HA-tagged and TLR4 is present, α VP3 represents an endogenous VP3 antibody, VP3 represents the amount of VP3 protein, and Con represents no load.

Con in FIG. 3 indicates the unloaded pCDNA3.1 expressed and TLR4 indicates the over-expression vector pCDNA3.1-HA-TLR 4.

EV in FIG. 5 represents the over-expression empty vector pCDNA3.1, and TLR4 represents the over-expression vector pCDNA3.1-HA-TLR 4.

Coni in FIG. 6 indicates knocked-out eGFP-unloaded PAM cells (control), TLR4-RNAi #1 indicates knocked-out TLR4 cell line.

coni in FIG. 8 indicates knocked-out eGFP-unloaded PAM cells (control), TLR4-RNAi #1 indicates knocked-out TLR4 cell line.

Preservation information:

Preservation time: 24 months 07 in 2019;

The name of the depository: china center for type culture Collection;

The preservation number is: CCTCC NO: c2019174;

The address of the depository: wuhan university in Wuhan, China;

And (3) classification and naming: the porcine alveolar macrophage TLR 4-1.

Detailed Description

the following examples are intended to better illustrate the technical solutions of the present invention, but not to limit the scope of the present invention.

FMDV used in the examples was provided by foot and mouth disease Virus reference laboratory, national institute of veterinary medicine, Lanzhou, national institute of agricultural sciences, Inc., and the lentiviral vector pGL-U6-gRNA was given by professor Suhong Dynasty, university of Wuhan, publicly available from NTCC type culture Collection, and Trans5 α competent cells, 293T cells, HEK-293T cells, PK-15 cells, and PAM cells were all available from Ribo Lai Biotech, Inc., Lanzhou.

Example 1 construction of TLR4 Gene knockout PAM cell line

1.1 design of sgRNA

determining an exon sequence according to a porcine TLR4 Gene sequence (Gene ID:399541), selecting a knockout site according to the exon sequence structure, and designing three pairs of sgRNA primers by using software, wherein the three pairs of sgRNA primers respectively comprise:

sgRNA-F-1:5’-CACCGGCCAGGACGAAGACTGGGTG-3’(SEQ ID NO.1)

sgRNA-R-1:5’-AAACCACCCAGTCTTCGTCCTGGCC-3’(SEQ ID NO.2)；

sgRNA-F-2:5’-CACCGGGGAGGACAGCGTCCTGGGG-3’(SEQ ID NO.3)

sgRNA-R-2:5’-AAACCCCCAGGACGCTGTCCTCCCC-3’(SEQ ID NO.4)；

sgRNA-F-3:5’-CACCGGCAGGAATACCTACCTGGAG-3’(SEQ ID NO.5)

sgRNA-R-3:5’-AAACCTCCAGGTAGGTATTCCTGCC-3’(SEQ ID NO.6)。

1.2 vector construction

1.2.1 enzyme digestion of empty plasmids (pGL-U6-gRNA) and recovery of gels

Mu.g of plasmid was digested with the restriction enzyme BSmbi, 37 ℃,2 h:

TABLE 1 restriction enzyme BSmb1 digestion system

BSmbI	5μL
		lentiviral vectors	5μg
NEB buffer	10μL
		sterilized water	Make up to 50 μ L

The digested plasmid was gel extracted with the kit, gel purified, and eluted in an Elution Buffer.

1.2.2 sgRNA primer annealing reactions

Table 2 annealing sgRNA reaction system

sgRNA-F	1μL
		sgRNA-R	1μL
Annealing buffer	48μL

Setting a PCR program: 90 deg.C, 4min, 70 deg.C, 10min, 37 deg.C, 20min, 10 deg.C, 20 min.

1.2.3 plasmid ligation and transformation screening

TABLE 3 plasmid ligation reaction System

sgRNA	6μL
		Enzyme digestion vector	2μL
T4 ligase	1μL
		T4 buffer	1μL

The reaction system described in Table 3 was mixed well and incubated at room temperature for 1h to obtain recombinant plasmids.

1.2.4 transformation selection

And (3) transformation: to an EP tube containing 100. mu.L of Trans 5. alpha. competent cells, 10. mu.L of the recombinant plasmid was added and mixed well. The tubes were kept on ice for 30min and then placed in a circulating water bath at 42 ℃ to heat shock for 90 s. Taking out, and rapidly cooling in ice for 5 min. Adding 1mL of non-resistant LB liquid culture medium into each tube, and resuscitating the mixture for 30min at 37 ℃ and 220rpm with slow shaking. Centrifuging the recovered bacterial liquid at 6000rpm at room temperature for 3min, sucking 800 μ L of supernatant, fully suspending the residual supernatant and the precipitated bacteria, and then coating the suspension on a solid LB culture medium containing ampicillin. The plates were incubated overnight in a 37 ℃ incubator.

Screening: picking out positive clones, upgrading the particles, and then carrying out enzyme digestion identification, wherein a reaction system is as follows:

TABLE 4 digestion reaction System of recombinant plasmid

Recombinant plasmid	8.2μL
		EcoRI	0.4μL
KpnI	0.4μL
		10×buffer	1μL

The reaction system described in Table 4 was incubated at room temperature for 30min and subjected to gel electrophoresis. The result of electrophoresis is that 10000bp position is slow virus carrier, 300bp position is sgRNA, the result shows that the positive plasmid selected is recombination plasmid.

1.3 Lentiviral packaging

The constructed recombinant plasmid and the pST1374-Cas9-D10A plasmid (the mass ratio of the recombinant plasmid to the pST1374-Cas9-D10A plasmid is 1:1, and the pST1374-Cas9-D10A plasmid is purchased from Riboley Biotechnology Ltd, Lanzhou) are transfected into 293T cells by lipofectamine 3000 liposome, the medium is changed into 10% FBS and DMEM without double antibody after 8-12 h of transfection, the incubation is continued for 12-24 h, when the cytopathic effect reaches 70%, cell supernatant is collected and filtered by a filter membrane with the size of 0.22 mu m for standby.

1.4 infection of cells

The method comprises the steps of paving PAM cells on a 12-hole plate, adding 1mL of lentivirus, 1mL of 10% FBS, DMEM without double antibody and 1.5 mu Lpolybrene when the cells grow to 30%, repeatedly infecting the cells after infecting the cells for 8-12 h, digesting the cells after 12h, transferring the cells to a 6-hole plate, culturing the cells by using DMEM added with 1% puromycin, and taking half of the cells after the cells grow to be full to perform a WB (protein immunoblotting) experiment to verify whether the TLR4 gene is knocked down.

the results are shown in fig. 1, only sgRNA (1#) constructed by sgRNA-F-1 and sgRNA-R-1 among the three pairs of constructed sgrnas has significant knockdown efficiency, and the other two effects are not significant, so that the effect on foot-and-mouth disease virus is detected only by #1 later. Example 2 analysis of TLR4 degradation of FMDV structural protein VP3

2.1 Experimental procedures

(1) Spreading HEK-293T cells on a 12-hole plate, and co-transfecting a signal path molecule plasmid pCDNA3.1-HA-TLR4 (purchased from Riborley Biotech, Lanzhou) with 0 mu g, 2 mu g, 4 mu g and 6 mu g of pCDNA3.1-FLAG-VP3 plasmid (purchased from Riborley Biotech, Lanzhou) of FMDV and 1 mu g of the plasmid respectively when the cells grow to 60-80 percent; pCDNA3.1-HA-TLR4 was constructed by inserting TLR4 gene fragment between EcoR I and Xho I of pCDNA3.1-HA-N vector shown in FIG. 9, and pCDNA3.1-FLAG-VP3 was constructed by inserting VP3 gene fragment between EcoR I and Xho I of pCDNA3.1-3Xflag-C vector shown in FIG. 10.

(2) and (3) collecting a sample after 24h of transfection, washing the sample for 1-2 times by using cooled PBS, adding 100 mu L of SDS loading buffer for cracking, and detecting the expression condition of the VP3 protein by using a WB method.

2.2 results of the experiment

As shown in FIG. 2, the content of VP3 protein gradually decreased as the dose of pCDNA3.1-HA-TLR4 plasmid was gradually increased. Therefore, the signal pathway molecule TLR4 can degrade VP3 protein, and the result shows that TLR4 can degrade VP3 protein.

Example 3 Effect of overexpression of TLR4 on degradation of VP3 protein

3.1 FMDV-infected cells

3.1.1 Experimental procedures

(1) Spreading PK-15 cells on a 12-well plate, when the cells grow to 60-80%, carrying out lipofectamine 3000 liposome transfection on PRK-HA-TLR4(5 mu g) and a corresponding empty vector pCDNA3.1(5 mu g), and infecting FMDV (MOI is 0.1) at different time points (0,6 and 12h) after 18h of transfection;

(2) After sampling, Q-PCR is respectively carried out to detect the copy number of the 3D gene of FMDV, and TCID50 and WB are used to detect the content of VP3 protein.

Q-PCR reaction reagent: 2 × one step RT-PCR buffer III (12.5 μ L), Takara Ex Taq HS (0.5 μ L), Primer script RT Enzyme mix II (0.5 μ L), forward Primer (5 'ACTGGGTTTTACAAACCTGTGA-3C) (0.5 μ L), reverse Primer (5' GCGAGTCCTGCCACGGA-3C) (0.5 μ L), probe (5FAM-TCCTTTGCACGCCGTGGGAC-TAMRA- -3A) (1 μ L), RNA (2 μ L), H₂O(7.5μL)；

WB detection: the samples are respectively collected, SDS-loading buffer is added for cracking, and the expression of the VP3 protein is detected by a WB method.

TCID50 testAnd (3) measurement: collecting cell supernatant after FMDV infection is finished, and using DMEM culture medium to make virus continuously dilute by 10 times, namely 10^-1,10^-2… …. Fold of dilution was determined from approximate titer of virus. The culture medium in a 96-well plate with about 80% of cells fully spread is aspirated by a discharging gun, washed 2 times with PBS (the purpose is to remove serum, which affects the adsorption of viruses), and discarded; 100ul of each dilution was added to a 96 well plate and 8 replicates for each dilution were performed. The final volume was 200 ul. And a blank control, i.e., 200ul dmem medium, was set. Incubated at 37 ℃. Cytopathic effects were observed at time points and the number of cytopathic wells was recorded. The results were calculated by the Reed-Muench two-handed method.

3.1.2 results of the experiment

As shown in fig. 3: Q-PCR results indicate that PAM cells can be infected with FMDV, and as the time of viral infection increases, FMDV gene copy number decreases in PAM cells that overexpress TLR 4.

As shown in fig. 4: WB assay results show that with increasing viral infection time, VP3 protein levels decreased after TLR4 overexpression compared to empty load.

As shown in fig. 5: TCID50 assays showed a decrease in lethality in cells overexpressing TLR4 compared to empty vectors.

TLR4 is known to be a key receptor, and both infectious and non-infectious stimuli induce pro-inflammatory responses with a key role as an amplifier of inflammatory responses. Foot-and-mouth disease virus also causes the body to develop an inflammatory response, an inflammation-inducing virus (GuiBoxiang, ChenQin, HuChuanxia, ZhuCaihui, HeGuimei. effects of calceriol (1,25-dihydroxy-vitamin D3) on the inflammation-induced by H9N2 Fluevinavirus infection in human lung A549 epithelial cells and in microorganisms [ J ] Virology J, 2017, 14). However, when infected with foot and mouth disease virus, PAM cells overexpressing TLR4 inhibited replication of foot and mouth disease virus instead and did not promote replication of foot and mouth disease virus as expected.

Example 4 Effect of knocking down TLR4 on promoting increase of VP3 protein

4.1.1 Experimental procedure (1) PAM WT cells (wild-type PAM cells without knocking down TLR4) and the PAM knockdown TLR4 gene cells prepared in example 1 were plated in 12-well plates, 12h followed by FMDV (MOI ═ 0.1), samples were collected at 6h and 12h, respectively, and absolute quantitative Q-PCR experiments were performed to determine the copy number of FMDV 3D gene, TCID50 and WB detection.

Q-PCR reaction conditions: 42 ℃ for 15 min; 95 ℃ for 10 s; 30s at 55 ℃; 72 ℃ for 30 s; and (4) carrying out 40 cycles in step 2 to step 4.

TCID50 detects: collecting cell supernatant after FMDV infection is finished, and using DMEM culture medium to make virus continuously dilute by 10 times, namely 10^-1,10^-2… …. Fold of dilution was determined from approximate titer of virus. The culture medium in a 96-well plate with about 80% of cells fully spread is aspirated by a discharging gun, washed 2 times with PBS (the purpose is to remove serum, which affects the adsorption of viruses), and discarded; 100ul of each dilution was added to a 96 well plate and 8 replicates for each dilution were performed. The final volume was 200 ul. And a blank control, i.e., 200ul dmem medium, was set. Incubated at 37 ℃. Cytopathic effects were observed at time points and the number of cytopathic wells was recorded. The results were calculated by the Reed-Muench two-handed method.

4.1.2 results of the experiment

The Q-PCR results are shown in FIG. 6: the 3D gene copy number of FMDV in PAM cells knocked down by TLR4 is obviously higher than that of WT PAM cells no matter 6 hours or 12 hours after FMDV is infected with PAM cells, and the result shows that the FMDV infection capacity of PAM cells is obviously enhanced after TLR4 knocking down.

WB detection is shown in fig. 7: with increasing time of viral infection, protein content of VP3 increased after TLR4 knockdown compared to empty load.

TCID50 detection is shown in FIG. 8: knockdown of TLR4 cell lethality was increased compared to empty load.

The above embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications made based on the structure, characteristics and principles of the invention should be included in the claims of the present invention.

SEQUENCE LISTING

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

Construction method and application of <120> TLR4 gene knocked-down porcine alveolar macrophage cell line

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Claims

1. The TLR4 gene knocked-down porcine alveolar macrophage cell line is characterized in that the preservation number of the porcine alveolar macrophage cell line is as follows: CCTCC NO: C2019174.

2. The method for constructing the TLR4 gene-knocked-down porcine alveolar macrophage cell line according to claim 1, wherein the TLR4 gene-knocked-down cell line is obtained by transfecting porcine alveolar macrophages with recombinant lentiviruses containing sgRNAs and performing resistance screening; the sgRNA is obtained by annealing a nucleotide sequence shown in SEQ ID NO. 1-2.

3. The method for constructing the TLR4 gene knocked-down porcine alveolar macrophage cell line according to claim 2, which comprises the following steps:

Step 1: designing a sgRNA primer F and a sgRNA primer R according to a porcine TLR4 gene sequence; the nucleotide sequence of the sgRNA primer F is shown as SEQ ID NO.1, and the nucleotide sequence of the sgRNA primer R is shown as SEQ ID NO. 2;

And step 3: packaging the recombinant lentiviral vector obtained in the step 2;

4. The method for constructing the TLR4 gene knocked-down pig alveolar macrophage lineage according to claim 2, wherein a reaction system for annealing the sgRNA primer F and the sgRNA primer R is as follows: 1 mu L of sgRNA primer F, 1 mu L of sgRNA primer R and 48 mu L of annealing buffer; reaction procedure: 90 deg.C, 4min, 70 deg.C, 10min, 37 deg.C, 20min, 10 deg.C, 20 min.

5. A lentiviral vector for knocking down the expression of TLR4 gene, wherein the lentiviral vector comprises the sgRNA of claim 2 or 3.

6. The use of the TLR4 knocked-down gene porcine alveolar macrophage cell line according to any one of claims 1-3 in promoting foot and mouth disease virus replication.