CN117887765B - A lentivirus-mediated stable expression IRF2 cell line and its construction method and use - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a construction method of a lentivirus-mediated stable expression IRF2 cell line, which comprises the steps of inoculating lentivirus containing an IRF2 gene of a chicken to DF-1 cells, and screening to obtain DF-1 cells capable of expressing IRF2 proteins. The cell line can stably express IRF2 genes, and further in vitro experiments prove that the over-expression IRF2 has the effect of promoting proliferation of H9N2 AIV. Experimental results show that stable IRF2 expressing cell lines can significantly increase the proliferation titer of H9N2AIV on cells. Meanwhile, the invention also provides a construction method and application of the cell line.

Description

A lentivirus-mediated stable expression IRF2 cell line and its construction method and use

Technical Field

The present invention relates to the field of biotechnology, and in particular to a lentivirus-mediated stable IRF2 expression cell line and a construction method and use thereof.

Background technique

Avian influenza virus (AIV) is a single-stranded negative-strand segmented RNA virus. Avian influenza virus is divided into H subtype and N subtype according to the antigenic characteristics of hemagglutinin (HA) and neuraminidase (NA) on its surface. Currently, 16 specific HAs and 9 specific NAs have been found in poultry, named H1~H16 and N1~N9 respectively. Avian influenza virus can be divided into highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) according to its pathogenicity. Although H9N2 subtype AIV is LPAIV, it is widely spread around the world. Since its first report in 1994, H9N2 AIV has been prevalent in my country. In recent years, H9N2 AIV has gradually become one of the main subtypes of avian influenza prevalent in my country. After poultry are infected with H9N2 LPAIV, their susceptibility to secondary infection with other pathogens increases, which can lead to high mortality and huge economic losses. In addition, H9N2 AIV can spread across hosts to infect mammals and even humans without the need for an intermediate host. The H9N2 subtype avian influenza virus can also provide internal genes for strains such as H5N1, H5N6, and H7N9 that infect humans. Therefore, the prevention and control of H9N2 AIV is not only crucial to the poultry industry, but also has important public health significance.

Interferon regulatory factors (IRFs) are a family of transcription factors that regulate interferon expression. The IRF family is a downstream transcription factor of multiple pattern recognition receptors, involved in a variety of biological processes, and plays different roles in the development, differentiation, and apoptosis of immune cells. Interferon regulatory factors can positively and negatively regulate viral and cellular gene expression in response to a variety of extracellular signals. Nine IRF members have been found in mammals, each with a different role. Studies have found that the transcription factor IRF2 drives interferon-mediated CD8 ⁺ T cell exhaustion to limit anti-tumor immunity; other studies have shown that transient overexpression of IRF2 in fish can significantly promote the replication of spring viremia of carp virus (SVCV) and inhibit the expression of antiviral-related genes.

Prior art 1: "Study on the interaction between non-structural proteins of avian influenza virus and host-related proteins and pathological changes of infected chicken tissues", 2008, Huazhong Agricultural University, Li Jianli, doctoral dissertation; the paper made a speculative conclusion: in vivo and in vitro, there is interaction between NS1 protein and ISG12-2 and IRF2 proteins. The interaction between NS1 protein and ISG12-2 and IRF2 may inhibit certain IFN expression-related pathways, antagonize IFN expression, reduce the body's antiviral state, and facilitate the replication and spread of AIV.

Prior art 2: "The dual nature of overexpression of the irf2 gene in medaka during viral infection", by Jiang Zhengzheng et al., Journal of Fisheries of China, Issue 9, 2021, which observed that Olirf2 can significantly inhibit the activity of NF-κB and ISRE, indicating that Olirf2 may promote viral proliferation by inhibiting the natural immune response of cells. However, continuous overexpression of Olirf2 enhances the antiviral ability of cells and promotes the expression of interferon-related genes mx1, ifn and irf3. Therefore, Olirf2 has a dual effect of antiviral or proviral based on the different duration of expression.

From the above records, it can be seen that the transient expression of the IRF2 gene generally promotes viral proliferation, while the continuous overexpression of the IRF2 gene is likely to inhibit viral proliferation.

At the same time, the existing technology 1 suggests that the IRF2 gene can promote the replication and spread of AIV and may be related to the NS1 protein.

In the art, DF-1 is a relatively recommended cell line for propagation of AIV, and its performance is relatively outstanding among several cell lines. However, even if DF-1 is used for propagation of AIV, its titer still cannot reach the ideal level.

Summary of the invention

The purpose of the present invention is to provide a lentivirus-mediated stable expression IRF2 cell line, which can stably express the IRF2 gene, and further prove through in vitro experiments that overexpression of IRF2 has the effect of promoting the proliferation of H9N2 AIV. The experimental results show that the stable expression of IRF2 cell line can significantly increase the proliferation titer of H9N2 AIV on cells.

At the same time, the present invention also provides a method for constructing a cell line and a use thereof.

To achieve the above object, the present invention provides the following technical solutions:

A method for constructing a lentivirus-mediated stable IRF2 expression cell line comprises inoculating a lentivirus containing the chicken IRF2 gene into DF-1 cells, and obtaining DF-1 cells capable of expressing the IRF2 protein through screening.

In the above construction method, the method is specifically:

Step 1: Connect the linearized lentiviral expression vector to the IRF2 gene to obtain a plasmid containing the IRF2 gene;

Step 2: Transfect the plasmid into HEK293T cells and collect the lentivirus containing the IRF2 gene;

Step 3: Inoculate the lentivirus into DF-1 cells, and obtain DF-1 cells that can express IRF2 protein after screening.

In the above construction method, the nucleotide sequence of the chicken IRF2 gene is shown in SEQ ID NO.1.

In the above construction method, the lentiviral expression vector is pLV-CMV-MCS-EF1-Puro.

In the above construction method, in step 2, the plasmid, packaging plasmid psPAX2 and pMD2.G are co-transfected into HEK293T cells.

At the same time, the present invention also discloses a lentivirus-mediated stable IRF2 expression cell line, which is prepared by any of the above methods.

Finally, the present invention also discloses the use of the IRF2 cell line described above to propagate avian influenza virus.

Compared with the prior art, the present invention has the following beneficial effects:

The DF-1 cell line overexpressing the IRF2 gene was successfully constructed using the lentiviral packaging method. The virus titer of H9N2 avian influenza in the constructed DF-1-IRF2 was significantly increased compared to that in DF-1 cells, which is conducive to the replication of avian influenza virus. The constructed DF-1-IRF2 cells can be used as a candidate vaccine cell line for H9N2 avian influenza.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1A is a diagram showing the amplification results of the IRF2 target gene fragment amplified from DF-1 cells;

Figure 1B is a diagram of the plasmid structure of pLV-CMV-IRF2-EF1-Puro;

FIG2A is a graph showing changes in the expression of the IRF2 gene in DF-1 control and DF-1-IRF2 cell lines;

FIG2B is a graph showing the western blot detection results of protein samples of control group cells and DF-1-IRF2 cells;

FIG3 is a graph showing the absorbance values of DF-1-IRF2 cells and control group cells DF-1 at a wavelength of 450 nm;

FIG4A is a graph showing the PCR results after H9N2AIV infected DF-1 cells and DF-1-IRF2 polyclonal cell lines, respectively;

FIG4B is a graph showing the expression of H9N2 AIV NP protein in DF-1-IRF2 cells and control group cells DF-1;

FIG4C is a photograph of DF-1 cells and DF-1-IRF2 cell lines infected with H9N2 AIV;

FIG4D is a graph showing the TCID ₅₀ results after H9N2 AIV infected DF-1 cells and DF-1-IRF2 cell lines, respectively;

FIG5 is a test result showing the effect of pCAGGS-IRF2 overexpression vector on the expression of H9N2 AIV virus NP mRNA;

Fig. 6 is a map of plasmid pCAGGS;

FIG. 7 is a map of pCAGGS-IRF2.

Detailed ways

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Part I Test Materials

1.1 Cells, viruses and vectors

H9N2 AIV, HEK-293T cells, DF-1 cells, and LMH cells are all maintained by the National and Local Joint Engineering Laboratory for Zoonotic Disease Prevention and Control Preparations of South China Agricultural University.

The lentiviral vector pLV-CMV-MCS-EF1-Puro, packaging plasmids psPAX2 and pMD2.G were kindly donated by Professor Cheng Yuqiang of Shanghai Jiao Tong University. The above vectors and plasmids can be purchased from Addgene. In addition, the literature "Establishment of a lentiviral vector-mediated stable expression eIF5A cell line and its effect on PRRSV proliferation", Journal of Animal Husbandry and Veterinary Medicine, published on January 11, 2023, also has relevant records by Li Huawei et al.; DH5α competent cells are products of Nanjing Novezan Company.

1.1.2 Main reagents

Plasmid extraction kit, homologous recombination enzyme, fluorescence quantitative detection kit, and ECL luminescence kit were purchased from Nanjing Novozyme; restriction endonuclease BamhⅠ was purchased from NEB; DMEM cell culture medium was purchased from Gibco, USA; DNA gel recovery kit was purchased from OMEGA; puromycin was purchased from biosharp; Nulen PlusTransTM transfection reagent was purchased from Shanghai Nolai Biotechnology; lentivirus concentration kit, RIPA lysis buffer reverse transcription kit, and HRP-labeled goat anti-mouse IgG were purchased from Shanghai Biotech Biotechnology. AIV NP monoclonal antibody was purchased from Invitrogen; IRF2 protein monoclonal antibody was purchased from Proteintech. 5× loading buffer: SDS-PAGE protein loading buffer produced by New Cyme. IRF2 mouse monoclonal antibody was purchased from Protech; HRP-labeled goat anti-mouse IgG was purchased from Biotech. AIV NP protein was purchased from GeneTex.

Part II Synthesis of Lentivirus

2.1 Primer design and synthesis

According to the chicken IRF2 sequence published by NCBI, a pair of specific primers were designed for the IRF2 gene (Gene ID: 396115) using the multi-clone pLV-CMV-MCS-EF1-Puro restriction site of the lentiviral expression vector.

The nucleotide sequence of the IRF2 gene (SEQ ID NO.1) is:

ATGCCTGTCGAAAGAATGCGGATGCGCCCATGGCTGGAAGAACAAATAAATTCTAACACAATACCAGGGCTGAAATGGATAAATAAGGAGAAGAAGATTTTTCAGATTCCCTGGATGCATGCTGCAAGACATGGGTGGGATGTTGAAAAAGATGCTCCCTTATTTAGAAACTGGGCAATTCACACAGGAAAATACCAGTCAGGAGTAGATAAACCCGACCCAAAGACATGGAAGGCAAACTTCCGGTGTGCTATGAACTCT CTGCCTGATATAGAAGAAGTGAAGGACAAAAGTATAAAGAAAGGAAACAATGCCTTCAGGGTGTACCGGATGCTGCCATTATCTGAAAGACCATCCAAAAAAGGAAAAAAAACGAAGTCTGAGAAGGATGACAAGTTCAAACAAATTAAGCAAGAGCCAGTTGAATCATCTTTTGGGATTAATGGGCTAAATGATGTTACTTCTGACTATTTCCTGTCCTCCAGTATAAAAAATGAAGTTGACAGTACAGTGAACATTGTAG TTGTAGGACAGCCGCACCTTGATGGCAGCAGTGAGGAGCAGGTGATAGTTGCCAATCCTCCAGATGTTTGCCAGGTAGTAGAGGTGACAACAGAAAGCGATGAGCAGCCTCTCAGCATGAGCCAGCTGTACCCCCTACAGATCTCCCCTGTCTCGTCCTATGCAGAAAGTGAAACGACCGACAGCGTTCCAAGCGATGAAGAAAATGCAGAGGGACGACTTCATTGGCAGAAGAAAAACATTGAAGGCAAACAGTATCTCAG CAATCTGGGAATGCGGAACACTTCTCATATGCTTCCCAGCATGGCTACTTTTGTAGCCAACAAGCCTGACCTCCAGGTCACCATCAAAGAAGAAAGCTGCCCGCTGCCTTACAACAGCTCCTGGCCCCCTTTCCCAGACATCCCTCTGCCACAAGTAGTGTCCACAGCCTCCACTAGCAGCAGCCGGCCGGACCGCGAGACACGGGCCAGCGTCATCAAGAAAACGTCAGACATCACCCAGTCAAGAGTCAAGAGCTGCTAA

Primer sequences are shown in Table 1.

Table 1 Primers used for PCR and qPCR

2.2 Construction of IRF2 recombinant vector

The vector pLV-CMV-MCS-EF1-Puro was digested by BamhⅠ restriction endonuclease at 37°C, and the linearized vector was recovered by electrophoresis gel; the process conditions for single digestion were as follows: 1 μg of empty pLV-CMV-MCS-EF1-Puro, 1 μL of BamhⅠ, 10 μL of digestion buffer, and enzyme-free water were added to make up to 50 μL, and digestion was performed in a constant temperature metal bath at 37°C overnight.

According to the manufacturer's instructions, the linearized vector and the target gene (as shown in SEQ ID NO.1) were mixed in the corresponding ratio, incubated at 37°C for 30 min, and then stored at -20°C. The recombinant product was transformed into competent cells DH5α, then spread on ampicillin-resistant solid LB plates and cultured overnight, and a single clone colony was picked and shaken for culture, and the bacterial solution was sequenced. After the sequencing was correct, the plasmid pLV-CMV-IRF2-EF1-Puro was expanded and extracted, and stored in a -20°C refrigerator for subsequent experiments.

2.3 Construction and packaging of recombinant lentiviral vectors

HEK293T cells in the logarithmic growth phase were evenly inoculated in a 5 cm cell culture dish (the culture medium was DMEM medium containing 10% FBS). After the cells grew to a monolayer, the recombinant plasmids pLV-CMV-IRF2-EF1-Puro, psPAX2 and Pmd2.G (the mass ratio of pLV-CMV-IRF2-EF1-Puro, psPAX2 and Pmd2.G was 4:2:2) were co-transfected into HEK293T cells (cell density 80%-90%, and cells were plated using a culture medium without antibiotics). At the same time, cells transferred with the empty vector pLV-CMV-MCS-EF1-Puro were set up as a control group. After incubation for 4-6 hours, fresh culture medium was replaced, and the lentiviral supernatant was collected 72 hours after transfection. The lentiviral supernatant was concentrated using a lentiviral concentration kit and placed at -80°C for later use.

Part III Lentiviral transduction, cell screening and identification

3.1 Lentiviral transduction and cell screening

The well-growing DF-1 cells were inoculated into a 24-well cell plate (about 200,000 cells per well). When the growth density reached 90%, the lentivirus and polybrene (the corresponding volume of polybrene was added at a ratio of 1:1000, and the polybrene concentration was controlled to be 8 mg/ml) were mixed and inoculated into the DF-1 cells. After culturing for 24 hours, fresh culture medium (DMEM culture medium containing 10% FBS) was replaced and cultured for 96 hours. After the cells grew stably, the growth medium containing 5 μg/mL of puromycin was replaced for screening. The screening was terminated when all the cells in the control group (cells not transfected with lentivirus) died. After the preliminary screening of positive cells, the screened cell suspension was subcultured into a 96-well cell culture plate by limiting dilution method to ensure that there were only 0 to 1 cells in each well. The resistance screening growth medium was used for another 10 to 14 days. The single cell clone with good growth status was observed and picked and inoculated into a 24-well plate. After the cell wells were full, they were identified to obtain the DF-1-IRF2 cell line.

The IRF2 target gene fragment was amplified from DF-1 cells, and its size was 1047 bp, as shown in Figure 1A; sequencing results showed that the cloned sequence was completely consistent with the published chicken IRF2 sequence, and the amplified fragment without the signal peptide coding sequence was inserted into the pLV-CMV-MCS-EF1-Puro vector.

The plasmid structure is shown in Figure 1B.

3.2 Identification of IRF2-overexpressing cell lines

3.2.1 Real-time PCR detection of IRF2 mRNA levels

The cells of the control group and the DF-1-IRF2 cell line obtained by screening were collected, and the cell RNA was extracted and reverse transcribed using an RNA extraction kit. The obtained cDNA was used as a template for Real-time PCR detection. The sequences of the Real-time PCR detection primers GADPH and IRF2 are shown in Table 1. The reaction procedure was: 95°C for 30 s, 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s for 40 cycles, and finally 60°C for 1 min and 95°C for 15 s to complete the detection.

The control group cells (DF-1 control) and the IRF2 overexpressing cell line (DF-1-IRF2 cell line) were collected, the total cell RNA was extracted and reverse transcribed into cDNA, and the Real-time PCR method was used to detect the changes in the expression level of the IRF2 gene in the DF-1 control and DF-1-IRF2 cell lines.

As shown in Figure 2A, the relative mRNA expression level of the IRF2 gene in DF-1-IRF2 cells was significantly higher than that in the control group cells (P<0.05), indicating that the integration of the lentiviral vector into the host cell genome can significantly increase the expression of IRF2.

3.2.2 Western blot detection of IRF2 protein level

The screened cells were evenly passaged into 6-well plates, and wild-type DF-1 cells were set as control group cells. After the cells grew to 80% confluent, the cells were lysed on ice with RIPA lysis buffer pre-added with PMSF, and SDS-PAGE was performed after adding 5× loading buffer and treating at 100°C. The proteins on the gel were transferred to a PVDF membrane, blocked with 5% skimmed milk powder for 1 h, and then IRF2 mouse monoclonal antibody was used as the primary antibody (1:1000 dilution) and HRP-labeled goat anti-mouse IgG was used as the secondary antibody (1:5000 dilution) and incubated at 37°C for 1 h. Finally, ECL chemiluminescent reagent was used to develop the image.

Protein samples of control group cells and DF-1-IRF2 cells were collected and analyzed by western blot. As shown in Figure 2B, the expression of IRF2 protein in DF-1-IRF2 cells was significantly increased compared with that in the control group cells.

3.2.3 Effect of stable expression of IRF2 gene on DF-1 cell activity

The control group DF-1 cells and the screened cells were plated in the center of a 96-well plate at 10 ⁴ cells per well. After the cells grew to an appropriate density, 10 μL of CCK-8 solution was added to each well at different time points, such as 0 h, 6 h, 12 h, and 24 h. The plates were gently shaken to mix, and then incubated at 37 °C in an incubator for 1 to 4 h. The OD values of the cell wells were detected at 450 nm using an enzyme reader, and the data were recorded.

DF-1 cells and DF-1-IRF2 were inoculated into 96-well plates at a density of 10 ⁴ cells per well. When the cells reached an appropriate density, the absorbance at 450 nm was detected using the CCK8 kit at 0, 6, 12 and 24 h. It was found that DF-1-IRF2 cells did not show slow growth or cell death, and the cell activity was normal. The CCK-8 results showed that there was no significant difference in the absorbance at 450 nm between DF-1-IRF2 cells and the control group cells DF-1, indicating that the stable expression of the IRF2 gene had no significant effect on the proliferation of DF-1 cells (P>0.05) (Figure 3).

Part IV Effect of stable expression on proliferation of H9N2 subtype AIV

4.1 Effect of stable expression of IRF2 on NP mRNA expression of H9N2 subtype AIV

The screened cell lines and control group cells transformed with an empty vector were inoculated into 12-well plates, and after the cell density reached 80%, H9N2 subtype AIV was inoculated with MOI = 0.1. The cells were collected at 24h and 48h, RNA was extracted and reversed into cDNA, and the expression of H9N2 AIVNP gene was detected by Real-time PCR. The Real-time PCR detection method and reaction conditions of H9N2 AIV were the same as those in Part 3, and the primer information used is shown in Table 1.

DF-1 cells and DF-1-IRF2 polyclonal cell lines were infected with H9N2 AIV, respectively. Two comparison groups were set up at 24h and 48h. The real-time PCR results showed that the expression of viral NP mRNA in the DF-1-IRF2 cell line infected with H9N2 AIV was significantly increased compared with the control group (P<0.05), indicating that overexpression of chIRF2 in vitro can significantly promote the replication of H9N2 AIV.

4.2 Effect of stable expression of IRF2 on the expression of H9N2 subtype AIV NP protein

The Western blot test method was the same as in the third part, with AIV NP protein as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody, and ECL chemiluminescence was used to develop the color and observe the results; the control group cells DF-1 and the screened cells DF-1-IRF2 were evenly passaged to a 24-well plate, inoculated with H9N2 AIV at MOI = 0.1, and fixed with pre-cooled paraformaldehyde for 30 minutes after 36 hours, permeated with 0.25% Triton for 30 minutes, and blocked with 5% BSA for 1 hour. The cells were washed 3 times with PBS before each operation, and the cells were incubated with NP monoclonal antibody as the primary antibody and FITC-labeled goat anti-mouse IgG as the secondary antibody at 37°C for 1 hour, evenly covered with DAPI colorimetric solution, stained in the dark for 5 minutes, and the results were observed under a Nikon fluorescent inverted microscope.

Western blot detection showed that the expression level of H9N2 AIVNP protein was significantly higher than that of the control group (Figure 4B), indicating that overexpression of IRF2 in vitro can significantly promote the expression of H9N2 AIV NP protein.

4.3 Effect of stable expression of IRF2 on the titer of H9N2 subtype AIV

DF-1-IRF2 cell line and control cell line were inoculated into T25 culture flasks, and H9N2 AIV virus diluted by DEME was inoculated into the cells respectively. The cells were incubated in a _CO2 incubator for 1 hour, and the DMEM culture medium containing 2% FBS was replaced. The cytopathic effect (CPE) was observed daily. When the CPE reached 90%, the cells were frozen and thawed three times at -80℃, and the cell supernatant was collected. LMH cells were plated on 96-well cell culture plates. After the cells grew to a monolayer, the virus solution was diluted with ^10-1 to ^10-10 10 concentration gradients and inoculated into LMH cells respectively. After culturing at 37℃ for 3-5 days, the CPE was observed and recorded, and the virus _TCID50 value was calculated by the Reed-Muench method, and the virus titers of the two were compared.

DF-1 cells and DF-1-IRF2 cell lines were infected with H9N2 AIV, respectively. The TCID ₅₀ results showed that the H9N2 virus titer in the DF-1-IRF2 cell line was higher than that in the control group (P<0.05), indicating that overexpression of IRF2 in vitro can significantly promote the titer of H9N2 AIV (P<0.05).

Part V DF-1 cell line transiently expressing IRF2 gene

Using the eukaryotic expression plasmid pCAGGS-HA, the pCAGGS-IRF2 overexpression plasmid was transfected into DF-1 cells, and H9N2 AIV was inoculated at MOI = 0.1 after 24 hours, and the pCAGGS-HA empty plasmid was used as a control. After 24 hours of culture, the total RNA of the cells was extracted using an animal cell total RNA extraction kit, and the expression level of NP mRNA of H9N2 AIV in DF-1 cells was detected by qPCR. Figure 5 shows the test results of the effect of the pCAGGS-IRF2 overexpression vector on the expression of NP mRNA of H9N2 AIV virus.

Among them, the map of the eukaryotic expression plasmid pCAGGS-HA is shown in Figure 6;

The construction method of pCAGGS-IRF2 is as follows: using EcorⅠ and KpnⅠ as restriction sites, upstream primer PCAGGS-IRF2-F (see Table 2), downstream primer PCAGGS-IRF2-R (see Table 2), italic 15bp as homology arms, using DNA of chicken embryo fibroblasts as template, amplifying the target gene fragment with homology arms, double-digesting PCAGGS, using the Norwegian homologous recombination kit to homologously recombinant the restriction empty load with the target gene fragment, obtaining the recombinant plasmid pCAGGS-IRF2, screening and identifying, and sequencing the correct extracted plasmid. The map of the recombinant plasmid pCAGGS-IRF2 is shown in Figure 7, and the sequence of the recombinant plasmid pCAGGS-IRF2 is shown in SEQ ID NO.2.

Table 2 Primer sequence list

Result analysis:

1. The present invention verifies that the DF-1 cell line transiently expressing the IRF2 gene can promote viral proliferation;

2. The present invention verifies that the DF-1 cell line that continuously overexpresses the IRF2 gene can promote viral proliferation;

This is contrary to the conclusion in the prior art that cell lines that continuously overexpress the IRF2 gene will inhibit viral proliferation;

The DF-1 cell line overexpressing the IRF2 gene was successfully constructed using the lentiviral packaging method. This cell line can be stably propagated, creating a stable, reliable, usable, and efficient cell line for subsequent virus propagation. The constructed DF-1-IRF2 has a significantly increased virus titer for H9N2 avian influenza compared to DF-1 cells, which is conducive to the replication of avian influenza viruses. The constructed DF-1-IRF2 cells can be used as a candidate vaccine cell line for H9N2 avian influenza.

Claims (6)

1. A method for constructing a cell line stably expressing IRF2 mediated by a lentivirus, characterized in that a lentivirus containing the chicken IRF2 gene is inoculated into DF-1 cells, and DF-1 cells capable of overexpressing the IRF2 protein are obtained through screening; The nucleotide sequence of the chicken IRF2 gene is shown in SEQ ID NO.1. 2. The construction method according to claim 1, characterized in that the method specifically comprises: Step 1: Connect the linearized lentiviral expression vector to the IRF2 gene to obtain a plasmid containing the IRF2 gene; Step 2: Transfect the plasmid into HEK293T cells and collect the lentivirus containing the IRF2 gene; Step 3: Inoculate the lentivirus into DF-1 cells, and after screening, obtain DF-1 cells that can overexpress IRF2 protein. 3. The construction method according to claim 2, characterized in that the lentiviral expression vector is pLV-CMV-MCS-EF1-Puro. 4. The construction method according to claim 2 is characterized in that in step 2, the plasmid, packaging plasmid psPAX2 and pMD2.G are co-transfected into HEK293T cells. 5. A lentivirus-mediated stable IRF2 expression cell line, characterized in that it is prepared by the method according to any one of claims 1 to 4. 6. Use of the IRF2 cell line according to claim 5 for propagating avian influenza virus, wherein the avian influenza virus is an avian influenza virus of the H9N2 subtype.