CN107190024A - A kind of construction method of stable expression NS1 albuminous cells strain - Google Patents

Abstract

The invention relates to a method for constructing a cell line stably expressing NS1 protein. Insert the NS1 gene at the EcoR I multiple cloning site of the pLenti-CMV-EGFP-3Flag-PGK-Puro vector, and use the constructed pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro recombinant plasmid to co-transform with the viral packaging plasmid Infect 293T cells, cultivate, release virus, collect virus supernatant, and filter to obtain recombinant lentivirus solution. The packaged recombinant lentivirus was used to infect A549 cells, and through puromycin selection, immunofluorescence and Western blot identification, a cell line stably expressing NS1 protein was obtained. The invention utilizes a recombinant lentiviral system to prepare a cell strain capable of stably expressing the NS1 protein by using a recombinant lentiviral system, and the cell strain provides a good tool for studying the biological function of the NS1 protein.

Description

A method for constructing a cell line stably expressing NS1 protein

technical field

The invention belongs to the field of medical molecular biology. Specifically, it relates to a method for constructing a lung cancer cell line stably expressing NS1 protein (Non-Structural 1 Protein, NS1).

Background technique

Lentivirus vector is a gene therapy vector developed on the basis of type I human immunodeficiency virus. Different from general retroviral vectors, lentiviral vectors can effectively integrate foreign genes into host chromosomes to achieve persistent expression. The lentiviral expression vector contains the genetic information required for packaging, transfection, and stable integration. It is necessary to use the expression vector and the packaging plasmid to co-transfect the cells and package the virus in the cells. The packaged virus particles are secreted into the extracellular medium, and the supernatant obtained by centrifugation contains high-titer virus particles, which can be directly used for the infection of host cells. After the target gene enters the host cell through virus infection, it is integrated into the genome, thereby continuously expressing the target gene at a high level. In cell-related experimental operations, for some cells that are difficult to transfect or even impossible to transfect by conventional methods, lentivirus-mediated transfection can greatly improve the transduction efficiency of genes to achieve high-efficiency expression of target genes.

Avian influenza virus is a type A influenza virus belonging to the family Orthomyxoviridae, genus Influenzavirus. The genome of influenza A virus consists of 8 segments of single-stranded negative-sense RNA, encoding 11-12 proteins, including: HA, NA, M1, M2, NS1, NS2, NP, PB1, PB2, PA, and NP40 and PB1-F2. Among them, NS1 protein (non-structural protein 1) is encoded by the RNA of the 8th segment of influenza virus, contains 202-237 amino acids, and has a relative molecular weight of about 28kD, which varies among different virus strains. Recent structural analysis found that this is mainly related to its specific structure. NS1 protein consists of three parts: RNA binding domain (RNA binding domain, RBD), effector domain (effector domain, ED) and the linker region (linker). region, LR), the flexible and variable linking region can make the NS1 protein present different conformations, so as to complete the combination with different RNAs and proteins. The RNA-binding domain (RBD) of NS1 consists of 73 amino acids and is capable of binding heterologous RNA, including viral genomic RNA, the 5' untranslated region of viral mRNA, poly(A) RNA, and exogenous dsRNA; the C-terminal effector of NS1 Domain (ED) is the main domain for NS1 to interact with the host, and has the functions of blocking host mRNA splicing, polyadenylation and nuclear translocation.

NS1 protein is closely related to the toxicity of influenza virus and plays an important role in regulating the pathogenicity of influenza virus. The NS1 protein of influenza A virus has the function of affecting host cell apoptosis. The process of its influence on host cell apoptosis has not been fully elucidated. Existing studies have found that NS1 protein has dual functions of promoting apoptosis and inhibiting apoptosis. In the early stage of infection, NS1 protein inhibits host cell apoptosis by reducing the expression of IFN to inhibit the downstream reaction of IFN; at the same time, NS1 can also interact with PI3K to activate PI3K/AKt signaling pathway, thereby inhibiting JNK-dependent, Bax-mediated induced host cell apoptosis. NS1 can also affect cell apoptosis through many other ways. It has been reported that activated PKR plays an important role in apoptosis during virus infection, and NS1 protein can directly bind to and inhibit the pro-apoptotic response activated by PKR; the N-terminal RNA binding region of NS1 protein can bind to intracellular dsRNA, thereby Inhibit the OAS/RNaseL pathway that promotes apoptosis; some studies have found that NS1 can inhibit the activity of p53 regulators, thereby inhibiting p53-regulated apoptosis; NS1 directly interacts with hGBP1 (Human guanylate-binding protein 1), resulting in hGBP1 The reduced GTPase activity indirectly inhibits the apoptotic response. Although apoptosis is often regarded as an antiviral response of the host cell itself to limit viral replication, the role of apoptosis in influenza A virus-infected cells is not well understood.

Contents of the invention

The purpose of the present invention is to use a recombinant lentiviral system to prepare a cell line that fuses a tag protein and can stably express the NS1 protein, and the cell line provides a good tool for studying the biological function of the NS1 protein.

The technical scheme adopted in the present invention is: a method for constructing a cell line stably expressing NS1 protein, comprising the following steps:

1) Amplification of the NS1 gene;

2) Connect the NS1 gene to the vector pLenti-CMV-EGFP-3Flag-PGK-Puro to construct the recombinant lentiviral expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro;

3) Co-transfect 293T cells with the recombinant lentivirus expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro and the virus packaging plasmid. Virus CMV-GFP-L.V.;

4) The packaged recombinant lentivirus CMV-GFP-L.V. was transfected into A549 cells, the positive cell lines were screened by puromycin, and the positive cell lines were identified by immunofluorescence and Western blot to obtain cell lines stably expressing NS1 protein.

The above-mentioned method for constructing a cell line stably expressing NS1 protein, in step 1), using NS1-F and NS1-R as specific primers, adding a restriction site EcoR I, and using the plasmid pEGFP-N1-NS1 as template for PCR amplification; the sequences of the specific primers NS1-F and NS1-R are:

NS1-F: 5'CCGGAATTCATGGATTCCAACACTGTGT 3'

NS1-R: 5' CCGGAATTCCGAACTTTTGACTCAATTGT 3'

The above-mentioned method for constructing a cell line stably expressing NS1 protein, the PCR amplification system is:

PCR reaction program: 94°C for 3min, 94°C for 30s, 55°C for 30s, 72°C for 1min, 4°C for 30 cycles.

In the above-mentioned method for constructing a cell line stably expressing NS1 protein, in step 3), the viral packaging plasmids are pLP1, pLP2 and pLP.

The beneficial effects of the present invention are:

1. The present invention uses a recombinant lentivirus as a carrier, infects A549 human lung adenocarcinoma cells, and screens to obtain a cell line that can stably express NS1 protein. The NS1 protein is fused to express EGFP and FLAG tags, which facilitates the detection of NS1 protein expression.

2. The present invention establishes the A549 cell strain capable of stably expressing NS1 protein, which lays a solid foundation for further in-depth research on the biological characteristics of NS1 protein and the pathogenic mechanism of avian influenza virus.

3. The present invention constructs a tagged cell line overexpressing NS1 protein, which provides a good tool for the study of the biological characteristics and functions of NS1 protein, and is also expected to become an ideal model for the treatment of avian influenza.

Description of drawings

Figure 1 is a diagram of the double enzyme digestion results of pEGFP-N1-NS1.

Figure 2 is the vector digestion result of pLenti-CMV-EGFP-3Flag-PGK-Puro;

Among them, 1: vector fragments; 2: 1kb DNA ladder marker: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp.

Fig. 3 is a schematic diagram of the structure of the pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro expression vector.

Figure 4 is the immunofluorescence identification of A549, cell lines transfected with empty viral vectors, and NS1 stable expression strains;

Among them, a: A549; b: cell line transfected with empty virus vector; c: NS1 stable expression strain.

Fig. 5 is the Western blot identification of NS1 stable expression strain;

Among them, 1: Marker; 2: A549Blank; 3: A549H102; 4: A549H4438.

detailed description

(1) NS1 gene amplification

1. NS1 gene PCR amplification

According to the NS1 gene sequence in GeneBank (GenBank: AAT90838.1), use the software LSPrimer (http://ccsipb.lnu.edu.cn/primer/) to design the specific primers NS1-F and NS1-R of the NS1 gene, add enzyme Cutting site EcoR I, the sequences of specific primers NS1-F and NS1-R are:

NS1-F: 5'CCGGAATTCATGGATTCCAACACTGTGT 3'

NS1-R: 5' CCGGAATTCCGAACTTTTGACTCAATTGT 3'

Use the pEGFP-N1-NS1 plasmid stored in our laboratory as a template for PCR amplification. The PCR reaction system is:

PCR reaction program: 94°C for 3min, 94°C for 30s, 55°C for 30s, 72°C for 1min, 4°C for a total of 30 cycles.

2. DNA agarose gel electrophoresis

Prepare 1.5% agarose, and heat it in a microwave oven to completely dissolve the agarose particles; place the washed and dried gel plate horizontally on the workbench; mix well, fill the gel, and insert a comb to avoid air bubbles; wait for the gel to solidify , carefully pull out the comb; put the gel into the electrophoresis tank, add the electrophoresis buffer; load the PCR product, and electrophoresis at 110V for 30min.

3. PCR product gel recovery

DNA was recovered according to the instructions of the agarose gel DNA recovery kit from Tiangen Company.

(2) Construction of recombinant lentiviral expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro

1. Digestion of pLenti-CMV-EGFP-3Flag-PGK-Puro vector

The pLenti-CMV-EGFP-3Flag-PGK-Puro vector was digested with EcoRI, and the digested system was placed in a water bath at 37°C overnight for electrophoresis, and the correct target band was recovered from the gel. The enzyme digestion system is as follows:

2. The NS1 gene is connected to the pLenti-CMV-EGFP-3Flag-PGK-Puro vector

React the NS1 gene recovered from the gel in (1) with the digested vector pLenti-CMV-EGFP-3Flag-PGK-Puro overnight at 16°C to construct the recombinant lentiviral expression plasmid pLenti-CMV-NS1-EGFP- 3Flag-PGK-Puro. The T4 enzyme ligation system is as follows:

3. Plasmid transformation into competent Escherichia coli E.coli DH 5α

Take out E.coli DH 5α competent cells and insert into crushed ice to dissolve; add 10 μl of the constructed recombinant lentiviral expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro to 50 μl of competent cells, and gently rotate to mix , ice-bathed for 30 minutes; heat-shock Escherichia coli in a water bath at 42°C for 45 seconds, quickly transfer it to wet ice, and let it stand for 2 minutes; add 500 μl of SOC culture medium preheated at 37°C without antibiotics, and shake it in a 37°C incubator at 180 rpm Culture for 1h. Take 120 μl of transformed competent cells and smear it on the LB agar plate containing 100 μg/ml ampicillin, place the plate at room temperature until it dries, then place it upside down in an incubator at 37°C for overnight culture for 12-16 hours, and check each culture Whether there are colonies in the dish. Pick a single colony on the agar plate and inoculate it into 5 ml of ampicillin-resistant LB medium, and culture it on a shaker at 1800 rpm at 37°C overnight, and the culture time should not exceed 16 hours.

4. Molecular biological identification of transformants

The genome of the extracted transformed strain was used as a template for PCR detection, and the recovered product was sequenced. Part of the bacteria liquid with correct sequencing was frozen and stored at -80°C for long-term storage. The other part of the bacterial liquid extracts the plasmid.

5. Plasmid extraction of pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro recombinant strain

The recombinant strains with correct sequencing were subjected to plasmid extraction to obtain the pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro recombinant lentiviral expression plasmid. A260 and A280 were measured by NanoDrop, and the calculated nucleic acid purity and nucleic acid concentration were A260/280=1.92, respectively, and the concentration was 350 ng/μl.

(3) Acquisition of recombinant lentivirus CMV-GFP-L.V.

1. Packaging of recombinant lentivirus

The constructed recombinant lentiviral expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro and viral packaging helper plasmids pLP1, pLP2, pLP were co-transfected into 293T cells (transfected with pLenti-CMV-EGFP-3Flag-PGK- Puro empty viral vector as a control). 24 hours before transfection, digest the 293T cells in the logarithmic growth phase with trypsin, adjust the cell density to ⁵ ×105 cells in the medium containing 10% serum, re-seek 10ml in a 10cm cell culture dish, 37°C, 5% CO ₂ Cultivate in an incubator for 24 hours, and the cells can be used for transfection when the cell density reaches 90%-95%. The cell culture medium was replaced with serum-free medium 2 h before transfection.

Add 4 μg of the prepared pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro vector, 4 μg of viral packaging plasmids pLP1, pLP2, and pLP to a sterilized 2ml centrifuge tube, and 1.5ml of serum-free Opti-MEM medium Mix well and incubate at room temperature for 5 minutes. Mix 48μl FuGene HD reagent with 1.5ml Opti-MEM in another tube and incubate at room temperature for 5 minutes. Mix the above two solutions and incubate at room temperature for 20 minutes to form a DNA-FuGene HD transfection complex. The mixture was transferred to the culture medium of 293T cells and cultured at 37°C in a 5% CO ₂ cell incubator. After 24 hours, pour out the culture medium containing the transfection mixture, add 10 ml of cell culture medium containing 10% serum to each bottle of cells, and continue culturing for 24 hours at 37° C. in a 5% CO ₂ incubator. After 24 h, the complete medium without antibiotics was replaced to continue the culture.

2. Collection and concentration of recombinant lentivirus

After 48 hours of transfection, collect the culture medium containing the virus into a 50ml sterile centrifuge tube, and then add 10ml of fresh complete culture medium to the cells that can still produce toxin; collect the culture medium containing the virus again after 72 hours of transfection, and divide the The collected culture solution was centrifuged at 4°C, 3000rpm, for 15min. Filter the virus supernatant with a 0.45 μm filter membrane to remove cell debris, centrifuge the filtrate at 4°C, 50,000 × g for 90 min, remove the supernatant, add 100 μl of complete culture medium, resuspend slowly and fully with a 200 μl pipette tip, and divide the virus concentrate Store in virus tubes after packaging, and store at -80°C for a long time to obtain recombinant lentivirus CMV-GFP-L.V.

3. Titer determination of recombinant lentivirus

On the first day, inoculate ⁴ ×104 293T cells in each well of a 96-well plate, and make a 10-fold serial dilution in EP tubes on the second day. The dilution method is: prepare eight 1.5ml EP tubes for each virus, and add 297μl complete culture solution, add 33μl virus stock solution to the first tube, after mixing, pipette 30μl into the second tube and mix well. By analogy, do 8 dilutions (10-10 ^-6 ). Discard the original culture medium in the 96-well plate, repeat 3 wells for each dilution, add 100 μl of diluted virus solution to each well and mark it. On the fifth day, the fluorescent positive cells were counted with a fluorescent microscope. Count the number of fluorescent cells in the last two wells where fluorescence can be observed, calculate the sum of the total number in the three repeated wells and calculate the average, assuming A (the average number of fluorescent cells in the penultimate well where fluorescence can be seen) and B (average number of fluorescent cells in the penultimate visible fluorescent well). Calculation formula of lentivirus titer: virus titer (TU/ml)=(A+B×10)×1000/2/virus volume μl in well A. The calculated virus titer was 4.62×10 ⁷ TU/ml.

(4) Screening of stably transfected cell lines

1. Optimization of optimal conditions for CMV-GFP-L.V. virus infection A549

According to experiments, A549 cells are infected with lentivirus CMV-GFP-L.V., the infection efficiency is 60%-70% at MOI 10, MOI 20, and the infection efficiency is high at MOI 40, reaching more than 80%. Based on the premise of high infection efficiency, low MOI value, and low cytotoxicity, the optimal infection conditions: A549 cells were selected at MOI 20-40 for subsequent experiments.

2. Screening of stable transfected cell lines

Resuscitate and culture A549 cells until they are in good condition. Before transfection, A549 cells are inoculated into 24-well plates at 30% confluence. A549 cells are made into a cell suspension of 4×10 ⁴ cells/ml and are ready to be plated. Spread 500 μl per well, that is, 2×10 ⁴ cells/well, and spread a 24-well plate. After 12 to 20 hours, the virus was infected, the titer of the virus liquid was 4.62×10 ⁷ TU/ml, and the virus volume was 17.3 μl. Add 10 μl of 1mg/ml polybrene to each well, and the final polybrene concentration in the cell sample is 5 μg/ml. Change the medium after 12-20 hours of infection: discard the medium, and add 2ml of fresh medium to each well. After infecting the cells for 72 hours, kill the cells that are not effectively infected by adding and maintaining 2ug/ml puromycin; every 2-3 days, change the fresh medium with a final concentration of 2ug/ml puromycin; under an inverted microscope Observation, screening of subclones, selection of normal growth cell populations for subculture, and gradual expansion of culture; thus screening positive cell lines under the maintenance of puromycin drug.

3. Detection of positive cell lines

Positive cells were identified by immunofluorescence using a fluorescence microscope (see Figure 4). The total protein was extracted after the positive cells were expanded and cultured, and the FLAG monoclonal antibody was used as the primary antibody for Western blot identification (see Figure 5). Finally, A549 cell line stably transfected with NS1 gene was obtained.

Claims (4)

1. A construction method for stably expressing NS1 protein cell strain, is characterized in that, comprises the steps: 1) Amplification of the NS1 gene; 2) Connect the NS1 gene to the vector pLenti-CMV-EGFP-3Flag-PGK-Puro to construct the recombinant lentiviral expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro; 3) Co-transfect 293T cells with the recombinant lentivirus expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro and the virus packaging plasmid. Virus CMV-GFP-L.V.; 4) The packaged recombinant lentivirus CMV-GFP-L.V. was transfected into A549 cells, the positive cell lines were screened by puromycin, and the positive cell lines were identified by immunofluorescence and Western blot to obtain cell lines stably expressing NS1 protein. 2. the construction method of a kind of stably expressing NS1 protein cell line according to claim 1, is characterized in that, described step 1), with NS1-F and NS1-R as specific primers, add enzyme cutting site EcoR I uses the plasmid pEGFP-N1-NS1 as a template to carry out PCR amplification; the sequences of the specific primers NS1-F and NS1-R are: NS1-F: 5'CCGGAATTCATGGATTCCAACACTGTGT 3' NS1-R: 5' CCGGAATTCCGAACTTTTGACTCAATTGT 3'. 3. the construction method of a kind of stably expressing NS1 albumen cell line according to claim 2, is characterized in that, PCR amplification system is: PCR reaction program: 94°C for 3min, 94°C for 30s, 55°C for 30s, 72°C for 1min, 4°C for 30 cycles. 4. The method for constructing a cell line stably expressing NS1 protein according to claim 1, characterized in that, in step 3), the viral packaging plasmids are pLP1, pLP2 and pLP.