CN110577934B - Construction method and application of TLR4 gene knocked-down porcine alveolar macrophage cell line - Google Patents

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 porcine alveolar macrophage cell line with knockdown of TLR4 gene

technical field

The invention relates to the field of biotechnology, in particular to a method for constructing a TLR4 gene knockdown porcine alveolar macrophage cell line and its application.

Background technique

Foot-and-mouth disease (FMD) is a highly contagious disease that mainly affects pigs, cattle, sheep, goats, deer and other cloven-hoofed animals. The causative agent of foot-and-mouth disease is foot-and-mouth disease virus (FMDV), which belongs to the foot-and-mouth disease virus genus of the Picornaviridae family. The viral genome contains a positive single-stranded RNA strand of 8.5 kb in length, encoding four structural proteins (VP1-VP4), eight non-structural proteins (L, 2A, 2B, 2C, 3A, 3B, 3C, and 3D) and some cleavage Intermediate. Viral proteins have multiple functions, enabling FMDV transmission and replication through many different mechanisms, and counteracting the host's antiviral response.

TLR is a type I transmembrane protein composed of an extracellular leucine-rich repeat domain, an intracellular conserved Toll/IL-1 receptor domain and a transmembrane domain. TLRs are the body's first line of immune defense against the invasion of external microorganisms, and are also a bridge connecting innate and adaptive immunity. They belong to the family of pattern recognition receptors. So far, 13 TLRs have been identified, which are widely distributed and mainly expressed in various immune cells, such as dendritic cells, macrophages, neutrophils, lymphocytes and microglia. TLRs can recognize microorganisms including bacteria, viruses and fungi, and can bind to their conserved sequences. Once the activation of TLRs is complete, a series of conserved cascades are initiated, culminating in the activation of two major intracellular transcription factors: NF-κB and interferon regulators.

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 constitute the first line of defense against infection. TLR4 has long been recognized as a receptor for Gram-negative lipopolysaccharide (LPS). In addition, it binds endogenous molecules produced by tissue damage. Thus, TLR4 is a key receptor that induces pro-inflammatory responses to both infectious and non-infectious stimuli. TLR4-mediated inflammation triggered by exogenous or endogenous ligands is also involved in several acute and chronic diseases, with a key role as an amplifier of the inflammatory response.

The gene of TLR4 is located on chromosome 9, mainly distributed in mononuclear macrophages, and can recognize bacterial lipopolysaccharide (LPS), bacterial teichoic acid and heat-shock proteins (HSP) released by host necrotic cells, etc. . The TLR4 gene was identified on the surface of human cells for the first time. It consists of three parts: the extracellular region, the transmembrane region and the intracellular region. It has a CDS sequence of 2526bp in full length and contains three exons. It can encode 841 genes in livestock. amino acid. After recent years of research, more biological functions of TLR4 have been found, including induction of immune inflammatory factors to trigger inflammatory responses, induction of specific immunity in livestock and poultry, assisting the body to clear viruses in the body, and synergistic effects with other TLRs molecules et al (MolteniMonica, GemmaSabrina, Rossetti Carlo. The Role of Toll-Like Receptor4 in Infectious and Noninfectious Inflammation. [J]. Mediators of inflammation, 2016, 2016.). Since most of its functions are involved in the immune response of the body, it is mainly expressed in monocytes, macrophages, granulocytes, dendritic cells, lymphocytes, epithelial cells, endothelial cells and bone marrow monocytes in livestock and poultry. host defense function in cells.

CRISPR-Cas9 gene editing technology is the third-generation genome editing technology developed rapidly after ZFN and TALEN technology. It is an adaptive immune defense formed by bacteria and archaea during the long-term evolution, which can be used to fight against invading viruses. and foreign DNA. CRISPR-Cas9 gene editing technology is a technology for specific DNA modification of targeted genes, and it has shown great application prospects in a series of gene therapy applications.

SUMMARY OF THE INVENTION

The invention utilizes the CRISPR-Cas9 gene editing technology to obtain a porcine alveolar macrophage cell line knocking down the TLR4 gene, and the obtained cell line can be used to promote the replication of foot-and-mouth disease virus.

The present invention specifically adopts the following technical solutions:

The present invention provides a porcine alveolar macrophage cell line with knockdown of TLR4 gene, and the cell line's deposit number is CCTCCNO: C2019174.

The invention also provides a method for constructing a TLR4 gene knockdown porcine alveolar macrophage cell line. The recombinant lentivirus containing sgRNA is transfected into porcine alveolar macrophage cells, and the TLR4 gene knockdown cell line is obtained through resistance screening; The sgRNA is obtained by annealing the nucleotide sequences shown in SEQ ID NO. 1-2.

Further, the construction method of the TLR4 gene knockdown porcine alveolar macrophage cell line specifically includes the following contents:

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

Step 2: digest the lentiviral vector, anneal the sgRNA primer F and the sgRNA primer R to obtain sgRNA, and connect the digested lentiviral vector and sgRNA, and the lentiviral vector is pGL-U6-gRNA;

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

Step 4: Infect porcine alveolar macrophages with the lentivirus obtained in step 3, and screen to obtain a TLR4 gene knockdown porcine alveolar macrophage cell line.

Further, the reaction system for the annealing of the sgRNA primer F and the sgRNA primer R is: sgRNA primer F 1 μL, sgRNA primer R 1 μL, annealing buffer 48 μL; 20min.

The present invention also provides a lentiviral vector for knocking down TLR4 gene expression, the lentiviral vector comprising the above-mentioned sgRNA.

The above-mentioned knockdown of TLR4 gene porcine alveolar macrophage cell line can be used to promote FMD virus replication.

The beneficial effects of the present invention are:

1. The present invention constructs a sgRNA primer pair for knocking down the TLR4 gene of PAM cells. Compared with the control primer pair, the knockdown efficiency of the primer pair is remarkable.

2. Compared with the cell line without knockdown, the replication amount of foot-and-mouth disease virus in the TLR4 gene knockdown porcine alveolar macrophage cell line prepared by the present invention is 2-3 times that of the former.

Description of drawings

Figure 1 shows the knockdown effect of sgRNA constructed by three pairs of sgRNA primers.

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

Figure 3 shows the 3D gene expression of FMDV after TLR4-overexpressing PAM cells were infected with FMDV.

Figure 4 shows the protein content of VP3 in the cells after TLR4-overexpressing PAM cells were infected with FMDV.

Figure 5 shows the detection results of TCID50 after PAM cells overexpressing TLR4 were infected with FMDV.

Figure 6 shows the 3D gene expression of FMDV in wild-type PAM cells and TLR4 knockdown PAM cells after FMDV infection of PAM cells.

Figure 7 shows the content of VP3 protein in wild-type PAM cells and TLR4-knockdown PAM cells after FMDV-infected PAM cells.

Figure 8 shows the TCID50 detection results of wild-type PAM cells and TLR4 knockdown PAM cells after FMDV infection of PAM cells.

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

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

In Figures 1, 2, 4 and 7, αTLR4 indicates the antibody to endogenous TLR4, αβ-actin indicates the antibody to the intracellular reference, αFlag indicates the Flag-tag antibody, αHA indicates the HA-tag antibody, and TLR4 indicates the endogenous The expression level of sexual TLR4, β-actin indicates the expression level of the cell internal reference, Flag-VP3 indicates that VP3 is Flag-tagged, indicates the content of VP3, HA-TLR4 indicates that TLR4 is HA-tagged, indicates the content of TLR4, and αVP3 indicates VP3 endogenous antibody, VP3 indicates the amount of VP3 protein, Con indicates empty load.

In Figure 3, Con represents the expressed empty vector pCDNA3.1, and TLR4 represents the overexpression vector pCDNA3.1-HA-TLR4.

In Figure 5, EV represents the overexpression empty vector pCDNA3.1, and TLR4 represents the overexpression vector pCDNA3.1-HA-TLR4.

In Figure 6, Coni represents the knockout eGFP empty PAM cells (control), and TLR4-RNAi#1 represents the knockout TLR4 cell line.

In Figure 8, Coni represents the knockout eGFP empty PAM cells (control), and TLR4-RNAi#1 represents the knockout TLR4 cell line.

Deposit information:

Preservation time: July 24, 2019;

Name of the depositary unit: China Type Culture Collection;

Deposit number: CCTCC NO: C2019174;

Depositary address: Wuhan University, Wuhan, China;

Classification name: porcine alveolar macrophage TLR4-1.

Detailed ways

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

The FMDV used in the examples was provided by the National Foot-and-Mouth Disease Virus Reference Laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The lentiviral vector pGL-U6-gRNA was donated by Professor Shu Hongbing of Wuhan University. The public can buy it from the NTCC Typical Culture Collection Center. Trans5α competent Cells, 293T cells, HEK-293T cells, PK-15 cells, and PAM cells were purchased from Lanzhou Ruibolai Biotechnology Co., Ltd.

Example 1 Construction of TLR4 gene knockout PAM cell line

1.1 Design of sgRNA

According to the porcine TLR4 gene sequence (Gene ID: 399541), determine the exon sequence, select the knockout site according to the exon sequence structure, and use the software to design three pairs of sgRNA primers, which are:

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 Empty plasmid (pGL-U6-gRNA) and gel recovery

Digest 5μg of plasmid with restriction enzyme BSmbI, 37℃, 2h:

Table 1 Restriction endonuclease BSmb1 digestion system

BSmbI 5μL Lentiviral vector 5μg NEB buffer 10μL Sterilized water Make up to 50 μL

Gel extraction kit digested plasmids, gel purified, and eluted in Elution Buffer.

1.2.2 sgRNA primer annealing reaction

Table 2 Annealing sgRNA reaction system

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

PCR program settings: 90°C, 4min, 70°C, 10min, 37°C, 20min, 10°C, 20min.

1.2.3 Plasmid ligation and transformation screening

Table 3 Plasmid ligation reaction system

sgRNA 6μL Restricted vector 2μL T4 ligase 1μL T4 buffer 1μL

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

1.2.4 Transformation screening

Transformation: Add 10 μL of recombinant plasmid to an EP tube containing 100 μL of Trans5α competent cells, and mix well. Place on ice for 30min, then place the EP tube into a 42°C circulating water bath for heat shock for 90s. Take out and quickly ice bath for 5min. Add 1 mL of anti-LB liquid medium to each tube, and recover with slow shaking at 37 °C and 220 rpm for 30 min. The recovered bacterial solution was centrifuged at 6000 rpm at room temperature for 3 min, 800 μL of the supernatant was removed, the remaining supernatant and the precipitated bacterial cells were fully suspended, and then coated on solid LB medium containing ampicillin. The plates were incubated overnight in a 37°C incubator.

Screening: Pick out positive clones, extract plasmids, and then carry out enzyme digestion identification. The reaction system is as follows:

Table 4 Recombinant plasmid digestion reaction system

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 30 min for gel electrophoresis. In the electrophoresis results, the lentiviral vector at 10000bp and the sgRNA at 300bp showed that the positive plasmids screened were recombinant plasmids.

1.3 Lentiviral Packaging

The constructed recombinant plasmid and pST1374-Cas9-D10A plasmid (the mass ratio of recombinant plasmid and pST1374-Cas9-D10A plasmid is 1:1, pST1374-Cas9-D10A plasmid was purchased from Lanzhou Ruibolai Biotechnology Co., Ltd.) and used lipofectamine 3000 liposomes were transfected into 293T cells. After 8-12 hours of transfection, the medium was changed to 10% FBS and DMEM without dual antibodies, and the incubation was continued for 12-24 hours. When the cytopathic effect reached 70%, the cell supernatant was removed. The supernatant was collected and filtered through a 0.22 μm filter until use.

1.4 Infecting cells

PAM cells were used to plate a 12-well plate, and when the cells grew to 30%, 1 mL of lentivirus, 1 mL of 10% FBS, DMEM without dual antibody, and 1.5 μL of polybrene were added, and the infection was repeated 8 to 12 hours later. After 12 hours, the cells were digested and transferred to 6 wells. and cultured in DMEM supplemented with 1% puromycin. After the cells were confluent, half of the cells were taken for WB (Western blotting) experiment to verify whether the TLR4 gene was knocked down.

The results are shown in Figure 1. Among the three pairs of sgRNAs constructed, only the sgRNA (1#) constructed by sgRNA-F-1 and sgRNA-R-1 had significant knockdown efficiency, and the other two had insignificant effects. 1# Detection of the impact of foot-and-mouth disease virus. Example 2 Analysis of TLR4 degradation of FMDV structural protein VP3

2.1 Experimental steps

(1) Spread HEK-293T cells into a 12-well plate, and when the cells grow to 60% to 80%, add the signaling pathway molecular plasmid pCDNA3.1-HA-TLR4 (purchased from Lanzhou Ruibolai Biotechnology Co., Ltd.) at 0 μg , 2μg, 4μg and 6μg were co-transfected into HEK-293T cells with 1μg of FMDV pCDNA3.1-FLAG-VP3 plasmid (purchased from Lanzhou Ruibolai Biotechnology Co., Ltd.); pCDNA3.1-HA-TLR4 was transfected by TLR4 The gene fragment was inserted between EcoR I and Xho I of the pCDNA3.1-HA-N vector shown in Figure 9, and pCDNA3.1-FLAG-VP3 was inserted into the pCDNA3.1- shown in Figure 10 by the VP3 gene fragment. It is constructed between EcoR I and Xho I of the 3Xflag-C vector.

(2) 24h after transfection, the samples were collected, washed 1-2 times with cold PBS, lysed by adding 100 μL of SDS loading buffer, and the expression of VP3 protein was detected by WB method.

2.2 Experimental results

The results are shown in Figure 2. As the dose of pCDNA3.1-HA-TLR4 plasmid gradually increased, the protein content of VP3 gradually decreased. Therefore, the above signaling pathway molecule TLR4 can degrade VP3 protein, indicating that TLR4 can degrade VP3 protein.

Example 3 Effect of overexpression of TLR4 on VP3 protein degradation

3.1 FMDV infected cells

3.1.1 Experimental steps

(1) Spread the PK-15 cells on a 12-well plate, and when the cells grow to 60% to 80%, use lipofectamine3000 liposome to transfect PRK-HA-TLR4 (5μg) and the corresponding empty vector pCDNA3.1 (5μg) ), FMDV was infected at different time points (0, 6, 12h) 18h after transfection (MOI=0.1);

(2) After the samples were collected, Q-PCR was performed to detect the copy number of FMDV 3D gene, TCID50 and WB to detect the content of VP3 protein.

Q-PCR reaction reagents: 2×one step RT-PCR bufferⅢ (12.5μL), Takara Ex Taq HS (0.5μL), Primer Scrip RT Enzyme mixⅡ (0.5μL), forward primer (5'ACTGGGTTTTACAAACCTGTGA-3C) (0.5 μL), reverse primer (5'GCGAGTCCTGCCACGGA-3C) (0.5 μL), probe (5FAM-TCCTTTGCACGCCGTGGGAC-TAMRA--3A) (1 μL), RNA (2 μL), H ₂ O (7.5 μL);

WB detection: Collect the above samples respectively, add SDS-loading buffer to cleave, and detect the expression of VP3 protein by WB method.

TCID50 detection: collect the cell supernatant after infection with FMDV, and use DMEM medium to serially dilute the virus 10 times, namely 10 ^-1 , 10 ^-2 , . . . Determine the dilution factor according to the approximate titer of the virus. Aspirate the culture medium in the 96-well plate with about 80% confluence of cells with a drain gun, and then wash twice with PBS (the purpose is to remove serum, because serum will affect the adsorption of viruses), discard; take 100ul for each dilution Add to 96-well plates with 8 replicates for each dilution. The final volume is 200ul. And set a blank control, namely 200ul DMEM medium. Incubate at 37°C. Cytopathies were observed by time point, and the number of cytopathic wells was recorded. The results were calculated using the Reed-Muench method.

3.1.2 Experimental results

As shown in Figure 3: Q-PCR results indicated that PAM cells could be infected with FMDV, and with the increase of virus infection time, the number of FMDV gene copies in PAM cells overexpressing TLR4 decreased.

As shown in Figure 4: WB detection results showed that with the increase of virus infection time, the content of VP3 protein after overexpression of TLR4 decreased compared with empty load.

As shown in Figure 5: TCID50 assay showed that the lethality of overexpressed TLR4 cells was lower than that of empty cells.

TLR4 is known to be a key receptor that induces pro-inflammatory responses by both infectious and non-infectious stimuli, with a key role as an amplifier of inflammatory responses. Foot-and-mouth disease virus can also cause an inflammatory response in the body and is an inflammation-inducing virus (GuiBoxiang, ChenQin, HuChuanxia, ZhuCaihui, HeGuimei. Effects of calcitriol (1,25-dihydroxy-vitamin D3) on the inflammatory response induced by H9N2 influenza virus infection in human lung A549 epithelial cells and in mice.[J].Virologyjournal,2017,14). However, when infected with FMD virus, PAM cells overexpressing TLR4 inhibited FMD virus replication instead of promoting FMD virus replication as expected.

Example 4 The effect of knocking down TLR4 on promoting the increase of VP3 protein

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

Q-PCR reaction reagents: 2×one step RT-PCR bufferⅢ (12.5μL), Takara Ex Taq HS (0.5μL), Primer Scrip RT Enzyme mixⅡ (0.5μL), forward primer (5'ACTGGGTTTTACAAACCTGTGA-3C) (0.5 μL), reverse primer (5'GCGAGTCCTGCCACGGA-3C) (0.5 μL), probe (5FAM-TCCTTTGCACGCCGTGGGAC-TAMRA--3A) (1 μL), RNA (2 μL), H ₂ O (7.5 μL);

Q-PCR reaction conditions: 42 °C, 15 min; 95 °C, 10 s; 55 °C, 30 s; 72 °C, 30 s; steps 2 to 4 for a total of 40 cycles.

WB detection: Collect the above samples respectively, add SDS-loading buffer to cleave, and detect the expression of VP3 protein by WB method.

TCID50 detection: collect the cell supernatant after infection with FMDV, and use DMEM medium to serially dilute the virus 10 times, namely 10 ^-1 , 10 ^-2 , . . . Determine the dilution factor according to the approximate titer of the virus. Aspirate the culture medium in the 96-well plate with about 80% confluence of cells with a drain gun, and then wash twice with PBS (the purpose is to remove serum, because serum will affect the adsorption of viruses), discard; take 100ul for each dilution Add to 96-well plates with 8 replicates for each dilution. The final volume is 200ul. And set a blank control, namely 200ul DMEM medium. Incubate at 37°C. Cytopathies were observed by time point, and the number of cytopathic wells was recorded. The results were calculated using the Reed-Muench method.

4.1.2 Experimental results

The results of Q-PCR are shown in Figure 6: No matter 6h or 12h after FMDV infection of PAM cells, the 3D gene copy number of FMDV in PAM cells with knockdown of TLR4 was significantly higher than that in WT PAM cells. The ability of FMDV to infect PAM cells was significantly enhanced after TLR4.

WB detection is shown in Figure 7: with the increase of virus infection time, the protein content of VP3 after knockdown of TLR4 increased compared with empty load.

TCID50 detection is shown in Figure 8: the lethality of knockdown TLR4 cells is higher than that of empty cells.

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> Construction method of porcine alveolar macrophage cell line with knockdown of TLR4 gene and its application

<130> None

<160> 6

<170> PatentIn version 3.5

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Claims (2)

1. A TLR4 gene knockdown porcine alveolar macrophage cell line, characterized in that the deposit number of the porcine alveolar macrophage cell line is: CCTCC NO: C2019174. 2. The application of the knockdown TLR4 gene porcine alveolar macrophage cell line according to claim 1 in promoting the replication of foot-and-mouth disease virus.

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