Application of BAG6 gene in regulation of influenza virus replication

Abstract

The invention discloses an application of a BAG6 gene in regulation of influenza virus replication. The invention provides any one of the following applications of the substance for inhibiting expression of the BAG6 gene or the encoded protein thereof: 1) preparing a product for expanding propagation of influenza virus; 2) preparing a product for promoting influenza virus replication; and 3) preparing a product for improving the expression quantity of the influenza virus related protein. Experiments prove that the function of the BAG6 gene encoding protein is lost, and the BAG6 gene encoding protein can be used for researching the cell biological function of the BAG6 protein and the action and mechanism in other viruses; in addition, the strain has the potential of being applied to influenza vaccine production. Besides, overexpression is carried out on the BAG6 protein, it is found that the BAG6 protein can inhibit the replication titer of the influenza virus and the expression of the protein, and the effect of the BAG6 protein in influenza virus replication is explored from the two aspects of knockout and overexpression.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to the application of BAG6 gene in regulating influenza virus replication. Background Art

[0002] CRISPR / Cas9 gene editing technology is currently the most advanced and effective gene editing technology, and is also one of the most popular research directions in the field of life sciences in recent years. Its core principle is to precisely cut the DNA sequence in the cell through a combination of a specific enzyme (Cas9) and a targeting RNA (crRNA) to modify, replace or delete the target gene. This technology has a wide range of application potentials and has been widely used in gene therapy, agriculture, biosafety and other fields.

[0003] In terms of gene therapy, CRISPR / Cas9 technology is used to treat a series of genetic diseases, such as cystic fibrosis and hemophilia. Through gene editing technology, defective genes in patients can be accurately repaired or replaced to achieve the purpose of treatment. In addition, CRISPR / Cas9 technology is also used in cancer treatment, by editing specific genes in cancer cells, thereby inhibiting the growth and spread of cancer cells. In the agricultural field, CRISPR / Cas9 technology can be used to improve crop varieties and increase crop yields and stress resistance. For example, by editing specific genes in crops, their adaptability to environmental factors such as drought and high temperature can be enhanced, thereby increasing crop yields and quality, while reducing environmental pollution. In addition, CRISPR / Cas9 gene editing technology also plays a vital role in studying gene and cell functions. By editing specific genes, the role and mechanism of the gene in the organism can be accurately studied. Therefore, CRISPR / Cas9 technology is one of the important tools for research in the field of life sciences, and is expected to bring more breakthroughs in human health and disease treatment.

[0004] Seasonal influenza is a common respiratory infectious disease, mainly caused by influenza A virus (IAV). Every year, seasonal influenza outbreaks pose a serious threat to public health and personal health worldwide. The hazards of seasonal influenza are mainly manifested in the following aspects: First, the seasonal influenza virus is highly contagious and can spread rapidly among the population through droplet transmission, leading to large-scale influenza outbreaks. Secondly, the severity of the patient's condition and the speed of transmission are very fast, which can easily cause social panic and anxiety. In addition, seasonal influenza can also pose a threat to individual health, especially for the elderly, children, pregnant women, and people with weak immune systems, the condition is more likely to worsen and even endanger life. Finally, due to the continuous mutation and evolution of seasonal influenza viruses, new variants of the virus and outbreaks of new influenza epidemics occur from time to time, causing long-term and far-reaching impacts on human health and public health security. At present, influenza vaccine production is still mainly based on chicken embryo production, but chicken embryo production has some limitations, such as long cycle, easy contamination of operation steps, lack of flexibility, and possible chicken embryo adaptive virus mutations; at the same time, chicken embryo production is also heavily dependent on the quantity and quality of chicken embryos, and some people are allergic to chicken embryos, which limits the production and use of vaccines. However, the advantages of cell production can make up for these, not relying on chicken embryos, and high flexibility. However, the current cell production system is not as mature as the chicken embryo production process, and it is necessary to continuously optimize the cell line itself, culture medium and process parameters, etc., to ultimately improve production efficiency and reduce production costs. Therefore, the construction of a cell line that promotes IAV replication through CRISPR-Cas9 technology is of great significance for the production of seasonal influenza vaccines.

[0005] BAG6 is a multifunctional host protein that participates in various cellular processes, such as protein quality control, apoptosis, and intracellular trafficking. Together with SGTA and ASNA1, BAG6 transports its ligand proteins to the endoplasmic reticulum for processing or post-translational modification. Ligand proteins that cannot be properly transported to the endoplasmic reticulum are ubiquitinated by RNF126, an E3 ubiquitin protein ligase associated with BAG6, and are sent to the proteasome for degradation. At the same time, BAG6 can bidirectionally regulate cell proliferation and apoptosis and has very important biological functions in vivo. However, the role of BAG6 in IAV infection is still unclear. Summary of the invention

[0006] The purpose of the present invention is to provide application of BAG6 gene in regulating influenza virus replication.

[0007] In a first aspect, the present invention provides the use of a substance for inhibiting the expression of the BAG6 gene or its encoded protein in any of the following:

[0008] 1) Preparation of expanded influenza virus products; expanded influenza viruses can also be used to prepare inactivated vaccines;

[0009] 2) Preparation of products that promote influenza virus replication;

[0010] 3) Preparation of products for increasing the expression of influenza virus-related proteins;

[0011] 4) Preparation of influenza virus for production of inactivated vaccines.

[0012] In the application described above, the influenza virus is influenza A virus.

[0013] In the application described above, the substance that inhibits the expression of the BAG6 gene or its encoded protein includes a CRISPR / Cas9 system that interferes with the expression of the BAG6 gene or its encoded protein.

[0014] The CRISPR / Cas9 system includes a plasmid expressing cas9 and a plasmid expressing sgRNA;

[0015] The targets of the sgRNA are the nucleotides shown at positions 10-33 of Sequence 1 and the nucleotides shown at positions 23-43 of Sequence 1.

[0016] In an embodiment of the present invention, the plasmids expressing sgRNA are plasmid target 1 and plasmid target 2.

[0017] The above-mentioned plasmid target 1 is a vector obtained by inserting the Human BAG6 target 1 sgRNA sequence into the Bbsl restriction site of the pUC19-sgRNA vector; wherein the Human BAG6 target 1 sgRNA sequence is obtained by connecting HumanBAG6 target 1-F and Human BAG6 target 1-R shown in Table 1 according to the PCR system shown in Table 2 and the PCR program shown in Table 3.

[0018] Plasmid target 2 is a vector obtained by inserting the Human BAG6 target 2 sgRNA sequence into the Bbsl restriction site of the pUC19-sgRNA vector; wherein the Human BAG6 target2 sgRNA sequence is obtained by connecting Human BAG6target 2-F and Human BAG6 target 2-R shown in Table 1 according to the PCR system shown in Table 2 and the PCR program shown in Table 3.

[0019] In an embodiment of the present invention, the influenza virus-related protein is exemplified by influenza virus NP protein.

[0020] In a second aspect, the present invention provides a recombinant cell for inhibiting the expression of BAG6 gene in a host cell to obtain a recombinant cell.

[0021] The recombinant cells described above are prepared according to a method comprising the following steps: introducing the CRISPR / Cas9 system described in claim 3 into the host cells to inhibit the expression of the BAG6 gene in the host cells.

[0022] In a third aspect, the present invention provides the use of the recombinant cell described in the second aspect in any of the following:

[0023] 1) Preparation and expansion of influenza virus products;

[0024] 2) Preparation of products that promote influenza virus replication;

[0025] 3) Prepare products for increasing the expression of influenza virus-related proteins.

[0026] In a fourth aspect, the present invention provides the use of the BAG6 gene or its encoded protein or a biological material related thereto or a substance for increasing the expression of the BAG6 gene or its encoded protein in any of the following:

[0027] 1) Preparing products for inhibiting influenza virus replication;

[0028] 2) Preparing products for inhibiting the expression of influenza virus-related proteins;

[0029] 3) Prepare anti-influenza virus products.

[0030] The above products are medicines.

[0031] In the application described above, the biological material related to the BAG6 gene is an expression cassette, a recombinant vector or a recombinant virus or a recombinant cell containing the BAG6 gene.

[0032] In an embodiment of the present invention, the recombinant vector is a BAG6-HA tag eukaryotic expression vector, specifically a vector obtained by replacing the fragment between the double restriction sites of BamHI and HindIII of the pRK5-HA vector (Biowind, the nucleotide sequence is sequence 3) with the BAG6 (human) gene sequence (sequence 1), and the BAG6 gene is fused with the HA tag on the vector to express the BAG6-HA protein.

[0033] In the application described above, the influenza virus is influenza A virus.

[0034] In order to clarify the role of BAG6 protein during influenza virus infection and evaluate the effect of its knockout on influenza virus replication, the present invention first provides two pairs of sgRNA targeting BAG6 gene, sgRNA can specifically target BAG6 gene, combined with CRISPR / Cas9 technology to achieve complete knockout of BAG6 gene in host cells, successfully constructed BAG6 gene knockout A549 cell line, accurate targeting, high knockout efficiency; CCK-8 kit was used to detect the growth activity of BAG6 gene knockout A549 cells and normal A549 cells, and the results showed no significant difference. Subsequently, a virus infection test was carried out in the BAG6 knockout cell line, and it was found that in the BAG6 knockout cells, the virus replication titer of influenza virus was significantly higher than that of normal cells, and the protein expression of the virus was also significantly increased in the BAG6 knockout cells. By making the function of the protein encoded by the BAG6 gene lost, it can be used to study the cell biological function of BAG6 protein and the role and mechanism in other viruses; and it has the potential to be applied to influenza vaccine production. In addition, the present invention also overexpressed the BAG6 protein and found that it could inhibit the replication titer of influenza virus and the expression of protein, and explored the role of BAG6 protein in influenza virus replication from the two aspects of knockout and overexpression. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a picture of single cell clusters observed after limiting dilution of transfected cells.

[0036] Figure 2 This is the result of Western blotting detection of BAG6 protein in BAG6 gene knockout A549 cells.

[0037] Figure 3 These are the results of the growth activity test of BAG6 gene knockout A549 cells and normal A549 cells.

[0038] Figure 4 The results of the growth and replication titer test of H1N1 virus in BAG6 gene knockout A549 cells.

[0039] Figure 5 These are the results of the growth and replication titer test of H7N9 virus in BAG6 gene knockout A549 cells.

[0040] Figure 6 These are the results of testing the growth and replication ability of H9N2 virus in BAG6 gene knockout A549 cells.

[0041] Figure 7 Western blotting results of viral protein expression of H1N1 virus in BAG6 gene knockout A549 cells.

[0042] Figure 8 The results of Western blotting detection of viral protein expression of H7N9 virus in BAG6 gene knockout A549 cells.

[0043] Fig. 9 The results of Western blotting detection of viral protein expression of H9N2 virus in BAG6 gene knockout A549 cells.

[0044] Fig.10 The results are the growth and replication titer detection results of H1N1 virus in A549 cells overexpressing BAG6 protein.

[0045] Fig.11 These are the results of growth and replication titer detection of H7N9 virus in A549 cells overexpressing BAG6 protein.

[0046] Fig.12 The results are the growth and replication titer detection results of H9N2 virus in A549 cells overexpressing BAG6 protein.

[0047] Fig.13 The results of Western blotting detection of viral protein expression of H1N1 virus in A549 cells overexpressing BAG6 protein.

[0048] Fig.14 These are the results of Western blotting detection of viral protein expression of H7N9 and H9N2 viruses in BAG6 protein-overexpressing A549 cells. DETAILED DESCRIPTION

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0050] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0051] The nucleotide sequence of the BAG6 gene in the following example is Sequence 1.

[0052] The pUC19-sgRNA vector (nucleotide sequence is sequence 2) in the following examples is described in the following literature: The name in the literature is pUC19 sgRNA; A simple and rapid approach to manipulate pseudorabiesvirus genome by CRISPR / Cas9 system, Biotechnol Lett. 2015Jun; 37(6): 1265-72.doi: 10.1007 / s10529-015-1796-2. Epub 2015Feb 28.

[0053] The cas9 plasmid in the following examples is described in the following literature: The name in the literature is pCDNA3.1 cas9; A simple and rapid approach to manipulate pseudorabies virus genome by CRISPR / Cas9 system, Biotechnol Lett. 2015Jun; 37(6): 1265-72.doi: 10.1007 / s10529-015-1796-2. Epub 2015Feb 28.

[0054] In the following examples, A / chicken / China / 0606-13 / 2017(H7N9) is recorded in the article Reassortment with Dominant Chicken H9N2 Influenza Virus Contributed to the Fifth H7N9 Virus Human Epidemic, and the name of the virus in the article is A / chicken / Shandong / M0303-10 / 2017(H7N9).

[0055] Example 1: Application of knocking out the BAG6 gene in promoting influenza virus replication

[0056] 1. Knockout of BAG6 gene by CRISPR / Cas9 system

[0057] (1) Design of sgRNA

[0058] The first 250 bases of the human BAG6 gene sequence were input into http: / / crispor.tefor.net / crispor.py, and appropriate targets were selected as guide sequences. Target 1 was nucleotides 10-33 of sequence 1, and target 2 was nucleotides 23-43 of sequence 1. The vector homology arm (4 bases) was added to the 5' end of the sequence, and the whole was used as the upstream primer. The downstream primer used the reverse sequence of the guide sequence plus the vector homology arm. Two pairs of primers shown in Table 1 were designed, with a distance of more than 100 bp between the primers.

[0059] Table 1

[0060]

[0061] (2) Construction of pUC19-sgRNA vector

[0062] The primers shown in Table 1 were dissolved to a final concentration of 100 μM, and the corresponding primers were subjected to the following PCR reaction to obtain double-stranded oligonucleotides (oligo, Human BAG6 target 1 or Human BAG6 target 2). The system and PCR program are shown in Tables 2 and 3 below:

[0063] ①PCR system:

[0064] Table 2

[0065]

[0066] ②PCR procedure:

[0067] Table 3

[0068]

[0069]

[0070] The pUC19-sgRNA vector was digested with enzymes and reacted in a 37°C water bath for 4 hours. The enzyme digestion system is shown in Table 4 below:

[0071] Table 4

[0072]

[0073] The digested vector was connected to the amplified oligo at room temperature for 2 hours. The connection system is shown in Table 5 below:

[0074] Table 5

[0075]

[0076] The ligation product was transferred into competent state, and after rejuvenation, it was plated and cultured overnight.

[0077] The bacterial solution PCR was used to identify whether the oligo was connected to the vector. The system and PCR procedure are shown in Tables 6 and 7 below:

[0078] ①PCR system:

[0079] Table 6

[0080]

[0081] ②PCR procedure:

[0082] Table 7

[0083]

[0084] The positive results were sequenced, and after sequencing was positive, the bacteria were shaken in large quantities to extract the plasmid.

[0085] The plasmid was extracted using the Plasmid Midiprep Purification Kit produced by Genemark.

[0086] Positive recombinant plasmid, the steps are as follows:

[0087] a. Take 20-100 mL of turbid bacterial solution, centrifuge at 12,000 × g for 5 min, and discard the supernatant.

[0088] b. Add 5 mL of Solution I, pipette to completely suspend the cell pellet, and let it stand at room temperature for 10 min.

[0089] c. Add 5 mL of Solution II and mix by inverting the centrifuge tube 6-8 times. The cell suspension will immediately become clear. Let it stand at room temperature for 2-4 minutes, but not more than 5 minutes.

[0090] d. Add 5 mL of Solution III and mix by inverting the tube 6-8 times.

[0091] e. Centrifuge the lysate at 12,000×g for 10 min at 4°C. A compact white precipitate should be visible on the side or bottom of the centrifuge tube.

[0092] f. Carefully transfer the supernatant to a new 50 mL centrifuge tube, add 10 mL of isopropanol to the centrifuge tube, and invert the centrifuge tube several times to mix the solution.

[0093] g. Centrifuge the mixture at 12,000×g for 10 min at 4°C. The white DNA precipitate will be at the bottom of the centrifuge tube. Discard the supernatant.

[0094] h. Add 500 μL of Binding Solution and gently pipette to completely suspend the DNA precipitate.

[0095] i. Place the Midi-spin Column in the Collection Tube, transfer the above DNA solution to the column, let it stand for 1 minute, and centrifuge at 12,000×g for 2 minutes.

[0096] j. Discard the filtrate, add 500 μL of Binding Solution, let stand for 2 min, and centrifuge at 12,000×g for 2 min.

[0097] k. Discard the filtrate, add 650 μL of Endotoxin Removal Wash Solution, let stand for 2 minutes, centrifuge at 12,000×g for 2 minutes. Discard the filtrate and repeat this step once.

[0098] l. Discard the filtrate, add 600 μL of Wash Solution, and centrifuge at 12,000×g for 2 min. Discard the filtrate and repeat this step twice.

[0099] m. Discard the filtrate and centrifuge at 12,000×g for 5 min.

[0100] n. Discard the Collection Tube and incubate the Midi-spin Column at 60-65℃ for 10 min to evaporate the residual ethanol.

[0101] o. Move the Midi-spin column to a new 1.5 mL centrifuge tube, add 150-300 μL of Elution Buffer preheated at 60-65°C to the column, let stand for 3 minutes, and centrifuge at 12,000×g for 2 minutes. The recovered plasmid can be stored at -20°C.

[0102] The following plasmid was obtained:

[0103] Plasmid target 1 is a vector obtained by inserting the Human BAG6 target 1sgRNA sequence into the Bbs1 restriction site of the pUC19-sgRNA vector; BAG6 target The 1sgRNA sequence was obtained by connecting Human BAG6target 1-F and Human BAG6 target 1-R shown in Table 1 according to the PCR system shown in Table 2 and the PCR program shown in Table 3.

[0104] Plasmid target 2 is a vector obtained by inserting the Human BAG6 target 2 sgRNA sequence into the Bbsl restriction site of the pUC19-sgRNA vector; wherein the Human BAG6 target2 sgRNA sequence is obtained by connecting Human BAG6target 2-F and Human BAG6 target 2-R shown in Table 1 according to the PCR system shown in Table 2 and the PCR program shown in Table 3.

[0105] (3) Construction and screening of BAG6 gene knockout cell lines

[0106] A549 cells in good condition (American type culture collection (ATCC), catalog number CRM-CCL-1856) were plated in a 6-well cell culture plate with a volume of 2 mL / well. When the cell confluence reached 70%-80%, transfection was prepared. 2 μg of cas9 plasmid, 1 μg of plasmid target 1 and 1 μg of plasmid target 2 were co-transfected into the 6-well culture plate of A549. The transfection process was as follows:

[0107] ① Add 200 μL jetPRIME Buffer (Polyplus, Catalog No. 101000046) to a 1.5 mL sterile centrifuge tube, add the corresponding volume of plasmid below the liquid surface (target 1 concentration 568 ng / μL, volume 1.76 μL; target2 concentration 628 ng / μL, volume 1.60 μL; cas9 concentration 708 ng / μL, volume 2.83 μL), shake to mix, and centrifuge instantly.

[0108] ② Add 8 μL of transfection reagent (total mass of 3 plasmids: 4 μg, transfection reagent volume = 1 ug: 2 μL), shake and mix, and then centrifuge instantly.

[0109] ③ Place the centrifuge tube at room temperature for 10 minutes. After the reaction is complete, gently drop the entire mixture into the 6-well plate cell culture medium (volume 2 mL), gently shake the cell plate to mix it with the cell culture medium. Place the cell plate in a 37°C, 5% CO2 incubator.

[0110] ④ 4 hours after transfection, remove the culture medium containing the transfection mixture and add 2 mL of fresh cell culture medium. Continue to culture in a 37°C, 5% CO2 incubator.

[0111] 24 h after transfection, cells were digested with 0.25% trypsin, 100-150 cells were counted, diluted with DMEM medium and evenly spread in two 96-well plates to ensure that most wells contained single cells.

[0112] After culturing for about 4-5 days, the cell status was observed, double-cell wells and multi-cell wells were removed, and the single-cell wells with normal cell morphology were marked; the culture was continued for about 7-9 days, and 4 monoclonal cell clusters were randomly selected for photographing and image collection.

[0113] Microscopic observation results Figure 1 As shown, the four figures are monoclonal cell clusters photographed from four different randomly selected cell wells, indicating that the monoclonal cell clusters are growing well and can be used for further culture and subsequent detection and analysis.

[0114] After confirming that the monoclonal cell clusters are growing well, the monoclonal cell clusters screened in the 96-well plate are digested with trypsin and transferred to 48-well plates for continued subculture. After the cells are fully grown, they are sequentially transferred to 24-well plates, 12-well plates, and 6-well plates for expansion culture.

[0115] After the monoclonal cells have grown to full size in the 6-well cell culture plate, each monoclonal cell is divided into two parts after trypsin digestion and transferred to a new 6-well plate for culture. One part is used for continued expansion culture, and the other part is used for Western blotting identification after it has grown to full size.

[0116] 2. Western blotting identification of BAG6 gene knockout A549 cell line

[0117] After the 6-well plate of the above 1 is full of cells, the cell samples are lysed with RIPA lysis buffer, and the cell samples are sonicated using a cell sonicator after instant centrifugation. The supernatant is collected by centrifugation, 6× protein loading buffer is added, and then the sample is placed in a 100°C metal bath and boiled for 10 minutes. The following experiment is performed:

[0118] (1) Glue preparation: Clean the glass plate, comb, glue strips, etc. in advance. Prepare 10% separation gel according to the formula, seal the gel surface with water, and after the separation gel solidifies, pour out the water and add 5% concentrated gel, insert a suitable comb, and after the gel solidifies, place it in the electrophoresis tank. Assemble the electrophoresis tank, fill the inner electrophoresis tank with the newly prepared electrophoresis solution, remove the comb, and fill the outer electrophoresis tank with the electrophoresis solution;

[0119] (2) Sample loading: Take 10 μL of the boiled supernatant prepared from the cells collected from each monoclonal cell well and put it into the sample loading well. In addition, add 10 μL of protein marker on both sides of the sample well.

[0120] (3) Electrophoresis: Set the electrophoresis instrument to run at a constant voltage of 65 V for 30 min, 120 V for 1 h, and 55 V. Pay attention to the electrophoresis until the bromophenol blue runs out of the bottom of the gel. When the electrophoresis is complete, gently remove the protein gel and rinse the gel with distilled water to remove the residual electrophoresis solution.

[0121] (4) Transfer: Soak the filter paper and sponge in precooled transfer solution in advance. After the electrophoresis is completed, carefully remove the gel block, refer to the marker on the protein gel, cut the gel according to the size of the target protein, and measure the length and width of the protein gel to cut the PVDF membrane of the appropriate size. Soak the PVDF membrane in anhydrous methanol for activation; in the transfer folder, lay out the sponge, 4 layers of filter paper, protein gel, PVDF membrane, 4 layers of filter paper, and sponge in this order. Note that each layer needs to be rid of bubbles with a glass rod. Finally, clamp the transfer folder and place it in the transfer tank. Pour in enough transfer solution and transfer at a constant current of 250 mA for 1 hour.

[0122] (5) Blocking: After the transfer is completed, remove the PVDF membrane and observe whether there is a clear marker band on the PVDF membrane to determine whether the transfer is successful. Place the membrane in 5% skim milk and block it on a horizontal shaker at room temperature for 1 hour or at 4°C overnight;

[0123] (6) Primary antibody incubation: After blocking, rinse the PVDF membrane quickly with PBST, place the clean membrane on BAG6 polyclonal antibody (Abnova, Catalog No. B01P) and β-actin monoclonal antibody (Sigma, Catalog No. A1978) diluted with primary antibody diluent, and incubate overnight on a horizontal shaker at 4°C;

[0124] (7) Secondary antibody incubation: After the primary antibody incubation is completed, the primary antibody is recovered, and the membrane is washed three times with PBST, each time for 5 min, and then 5% skim milk is added to dilute HRP-labeled IgG 10,000 times, and incubated on a horizontal shaker at room temperature for 1 h;

[0125] (8) Color development: After incubation with the secondary antibody, wash the membrane three times with PBST, 10 min each time, and turn on the Tanon 5200 fully automatic chemiluminescence image exposure instrument in advance. After washing, add an appropriate amount of color development solution to the PVDF membrane until the membrane surface is completely covered, and color development is carried out in the dark for 2 min;

[0126] (9) Exposure: Use tweezers to remove the PVDF membrane from the luminescent solution and place the membrane in an exposure instrument for exposure. The exposure time is determined according to the amount of protein expression. Save the exposure image and analyze the results.

[0127] The results are as follows Figure 2 As shown, WT is the A549 cell line, #1-#17 are the monoclonal cells obtained in 1 above, and it can be seen that compared with the A549 cell line, the expression level of BAG6 in #4 and #17 is reduced, proving that the BAG6 gene in the knockout cells is knocked out, and these cells are named BAG6 gene knockout A549 cell lines.

[0128] 3. CCK8 kit (Biyuntian, Cat. No. C0042) to detect cell activity

[0129] (1) A piece of BAG6 gene knockout A549 cell #17 (denoted as BAG6-KO in the figure) and a piece of normal A549 cell (denoted as WT in the figure) identified in 2 above were respectively plated in a 96-well plate, and approximately 2000 cells were added to each well by cell counting;

[0130] (2) Then, 10 μL of enhanced CCK-8 solution was added to each well at 12, 24, 36, and 48 h after plating.

[0131] (3) After adding CCK-8 solution at each time point, the cells were incubated in the cell culture incubator for 1 hour.

[0132] (4) Measure the absorbance at 450 nm.

[0133] The results are as follows Figure 3 As shown, there was no significant difference in the growth activity between BAG6 gene knockout A549 cells and normal A549 cells.

[0134] 4. Detection of influenza virus titer in BAG6 gene knockout A549 cells

[0135] (1) Normal A549 cells (denoted as WT in the figure) and BAG6 gene knockout A549 cells (denoted as BAG6-KO in the figure) were plated in 6-well cell culture plates, and the 6-well plates were placed in a 37°C, 5% CO2 incubator for culture.

[0136] (2) When the cells grew to a density of about 80%-90%, A / Puerto Rico / 8 / 1934(H1N1) (A / chicken / China / 0606-13 / 2017(H7N9) and A / chicken / Shandong / 196 / 2011(H9N2) were also used) were used to infect the above two types of cells at an MOI of 1. After the virus was adsorbed for 1 hour, the virus infection solution was discarded, the plate was gently washed three times with PBS, and DMEM medium containing TPCK-trypsin and 1% double antibody was added, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0137] (3) Collect 100 μL of cell supernatant at 6 h, 12 h, 24 h, and 36 h after infection (the sampling time for H7N9 and H9N2 virus infections is 12 and 24 h after infection) and mark each tube; temporarily store the cell supernatant in a -80 °C refrigerator for subsequent unified use.

[0138] (4) One day in advance, MDCK cells were plated in a 96-well plate. When the cell density reached 70%-80%, the serum-containing DMEM medium was discarded and the cells were washed three times with PBS buffer.

[0139] (5) Take out the frozen virus solution from the -80℃ refrigerator 30 minutes in advance and place it on ice to thaw naturally. Prepare 8 1.5mL sterile centrifuge tubes and add 450μL of DMEM containing TPCK-trypsin (final concentration of 2μg / mL) and 1% double antibody to each tube. Add 50μL of the virus stock solution to the first tube and shake to mix, aspirate 50μL of the mixture and add it to the second tube, dilute it tenfold until the 8th tube, mark it, and temporarily place it on ice.

[0140] (6) Virus inoculation The virus of each dilution was inoculated into a 96-well plate at a dose of 100 μL / well, and three parallel replicates were performed for each dilution. After culturing for about 36-48 hours, the following indirect immunofluorescence (IFA) test was performed.

[0141] (7) IFA test

[0142] ① Washing: Aspirate the cell culture medium, wash the cells once with PBS buffer, and discard the washing solution.

[0143] ② Fixation: Prepare the fixative solution in the ratio of ethanol:acetone = 3:2. Add the pre-cooled fixative solution to the cell wells, 50 μL / well, and let it stand at room temperature for 15 minutes.

[0144] ③Washing: Aspirate the fixative and wash the cells 3 times with PBS buffer.

[0145] ④ Primary antibody incubation: Dilute influenza virus NP protein monoclonal antibody (Abcam, catalog number AB20343) 5000 times with PBS, add 30 μL to each well, and incubate the reaction in a 37°C incubator for 1 hour (or 4°C overnight).

[0146] ⑤Washing: Remove the primary antibody in the well, wash the cells with PBS buffer, and repeat the washing 3 times.

[0147] ⑥ Secondary antibody incubation: Dilute FITC-labeled goat anti-mouse IgG (SeraCare (KPL), catalog number 5230-0427) 400 times with PBS, add 30 μL to each well, and incubate in a 37°C incubator in the dark for 1 hour.

[0148] ⑦ Washing: Remove the secondary antibody in the wells and wash the cells with PBST (add 0.5 mL Tween-20 to 1000 mL PBS). Wash on a shaker for 3 minutes after adding the washing solution each time and then discard the washing solution. Repeat the washing three times.

[0149] ⑧Result observation: Cells that emit green fluorescence under a fluorescence microscope are considered to be virus-infected positive cells. TCID is calculated according to the Reed-Muench method 50 .

[0150] The results are as follows Figure 4-Figure 6As shown, it can be seen that the viral replication titer of influenza virus in BAG6 gene knockout A549 cells is significantly higher than that in normal A549 cells.

[0151] 5. Detection of viral protein expression of influenza virus in BAG6 gene knockout A549 cell line

[0152] (1) Normal A549 cells (denoted as BAG6-WT in the figure) and BAG6 gene knockout A549 cells (denoted as BAG6-KO in the figure) were plated in 6-well cell culture plates, and the 6-well plates were placed in a 37°C, 5% CO2 incubator for culture.

[0153] (2) When the cells grew to a density of about 80%-90%, A / Puerto Rico / 8 / 1934(H1N1) (A / chicken / China / 0606-13 / 2017(H7N9) and A / chicken / Shandong / 196 / 2011(H9N2) were also used) were used to infect the above two types of cells at an MOI of 1. After the virus was adsorbed for 1 hour, the virus infection solution was discarded, the plate was gently washed three times with PBS, and DMEM medium containing TPCK-trypsin and 1% double antibody was added, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0154] (3) At 6 h, 12 h, 24 h, and 36 h after infection (the sampling time for H7N9 and H9N2 virus infections was 12 and 24 h after infection), the cell samples were lysed using RIPA lysis buffer, and after instant centrifugation, the cell samples were sonicated using a cell sonicator. The supernatant was collected by centrifugation, 6× protein loading buffer was added, and then the samples were boiled in a 100°C metal bath for 10 min.

[0155] (4) The expression of viral proteins was detected by Western blotting method as described in 2 above. The primary antibodies were BAG6 polyclonal antibody, NP polyclonal antibody (GenScript, catalog number A01506-40) and β-actin monoclonal antibody.

[0156] The results are as follows Figure 7-Figure 9 As shown, hpi is hour post-infection time (h); compared with normal cells A549, the expression of viral NP protein was also significantly increased in BAG6 gene knockout A549 cells.

[0157] The above results indicate that inhibiting BAG6 gene expression or inhibiting BAG6 protein activity can enhance the replication ability of influenza virus, enable influenza virus propagation, and have the potential to be used in influenza vaccine production.

[0158] Example 2: Application of overexpressing BAG6 gene in inhibiting influenza virus replication

[0159] 1. Construction of BAG6-HA tag eukaryotic expression vector

[0160] The BAG6-HA tag eukaryotic expression vector is a vector obtained by replacing the fragment between the double restriction sites of BamHI and HindIII of the pRK5-HA vector (Biowind, the nucleotide sequence is sequence 3) with the BAG6 (human) gene sequence (sequence 1). The BAG6 gene is fused with the HA tag on the vector to form the BAG6-HA protein.

[0161] 2. Detection of influenza virus titer in BAG6 protein overexpressing A549 cells

[0162] (1) A549 cells in good condition were plated in a 6-well cell culture plate, with 2 mL per well, and the 6-well plate was then placed in a 37° C., 5% CO 2 incubator for culture.

[0163] (2) When the cells grow to a 60%-70% fusion state, 1.5 μg of pRK5-HA empty vector (denoted as EV in the figure, concentration is 534 ng / μL, transfection volume is 2.81 μL) and BAG6-HA tag eukaryotic expression vector (denoted as BAG6-HA in the figure, concentration is 664 ng / μL, transfection volume is 2.26 μL) are transfected respectively. The transfection method is the same as described in (3) of Example 1. 24 h after transfection, the above cells were infected with A / Puerto Rico / 8 / 1934(H1N1) (A / chicken / China / 0606-13 / 2017(H7N9) and A / chicken / Shandong / 196 / 2011(H9N2) were also used) at MOI=1. After 1 h of virus adsorption, the virus infection solution was discarded, the plate was gently washed three times with PBS, and DMEM medium containing TPCK-trypsin and 1% double antibody was added, and cultured in a 37°C, 5% CO2 incubator.

[0164] (3) Collect 100 μL of cell supernatant at 12 h, 24 h, and 36 h after infection (the sampling time for H7N9 and H9N2 virus infections is 12 and 24 h after infection) and mark each tube; temporarily store the cell supernatant in a -80 °C refrigerator for subsequent unified use.

[0165] (4) One day in advance, MDCK cells were plated in a 96-well plate. When the cell density reached 70%-80%, the serum-containing DMEM medium was discarded and the cells were washed three times with PBS buffer.

[0166] (5) Take out the frozen virus solution from the -80℃ refrigerator 30 minutes in advance and place it on ice to thaw naturally. Prepare 8 1.5mL sterile centrifuge tubes and add 450μL of DMEM containing TPCK-trypsin (final concentration of 2μg / mL) and 1% double antibody to each tube. Add 50μL of the virus stock solution to the first tube and shake to mix, aspirate 50μL of the mixture and add it to the second tube, dilute it tenfold until the 8th tube, mark it, and temporarily place it on ice.

[0167] (6) Virus inoculation The virus of each dilution was inoculated into a 96-well plate at a dose of 100 μL / well, and three parallel replicates were performed for each dilution. After culturing for about 36-48 hours, the following indirect immunofluorescence (IFA) test was performed.

[0168] (7) IFA test

[0169] ① Washing: Aspirate the cell culture medium, wash the cells once with PBS buffer, and discard the washing solution.

[0170] ② Fixation: Prepare the fixative solution in the ratio of ethanol:acetone = 3:2. Add the pre-cooled fixative solution to the cell wells, 50 μL / well, and let it stand at room temperature for 15 minutes.

[0171] ③Washing: Aspirate the fixative and wash the cells 3 times with PBS buffer.

[0172] ④ Primary antibody incubation: Dilute the influenza virus NP protein monoclonal antibody 5000 times with PBS, add 30 μL to each well, and incubate the reaction in a 37°C incubator for 1 hour (or 4°C overnight).

[0173] ⑤Washing: Remove the primary antibody in the well, wash the cells with PBS buffer, and repeat the washing 3 times.

[0174] ⑥ Secondary antibody incubation: dilute FITC-labeled goat anti-mouse IgG 400 times with PBS, add 30 μL to each well, and incubate in a 37°C incubator in the dark for 1 hour.

[0175] ⑦ Washing: Remove the secondary antibody in the wells and wash the cells with PBST (add 0.5 mL Tween-20 to 1000 mL PBS). Wash on a shaker for 3 minutes after adding the washing solution each time and then discard the washing solution. Repeat the washing three times.

[0176] ⑧Result observation: Cells that emit green fluorescence under a fluorescence microscope are considered to be virus-infected positive cells. TCID is calculated according to the Reed-Muench method 50 .

[0177] The results are as follows Figure 10-12As shown, it can be seen that in A549 cells overexpressing BAG6 protein (denoted as BAG6-HA in the figure), the viral replication titer of influenza virus is significantly lower than that in A549 cells transfected with pRK5-HA empty vector (denoted as EV in the figure).

[0178] 3. Detection of viral protein expression of influenza virus in A549 cell line overexpressing BAG6 protein

[0179] (1) A549 cells in good condition were plated in a 6-well cell culture plate, with 2 mL per well, and the 6-well plate was then placed in a 37° C., 5% CO 2 incubator for culture.

[0180] (2) When the cells grew to a 60%-70% fusion state, 1.5 μg of pRK5-HA empty vector (denoted as EV in the figure, concentration is 534 ng / μL, transfection volume is 2.81 μL) and BAG6-HA tag eukaryotic expression vector (denoted as BAG6-HA in the figure, concentration is 664 ng / μL, transfection volume is 2.26 μL) were transfected respectively, and the transfection method was the same as above.

[0181] (3) 24 h after transfection, the two cell types were infected with A / Puerto Rico / 8 / 1934(H1N1) (A / chicken / China / 0606-13 / 2017(H7N9) and A / chicken / Shandong / 196 / 2011(H9N2) were also used) at an MOI of 1. After 1 h of virus adsorption, the virus infection solution was discarded, the plate was gently washed three times with PBS, and DMEM medium containing TPCK-trypsin and 1% double antibody was added, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0182] (3) At 6 h, 12 h, 24 h, 36 h, and 48 h after infection (the sampling time for H7N9 and H9N2 virus infections was 12 and 24 h after infection), the cell samples were lysed using RIPA lysis buffer, and after instant centrifugation, the cell samples were sonicated using a cell sonicator. The supernatant was collected by centrifugation, 6× protein loading buffer was added, and then the samples were boiled in a 100°C metal bath for 10 min.

[0183] (4) The expression of viral proteins was detected by Western blotting method as described in 2 above. The primary antibodies were HA tag monoclonal antibody (Sigma, catalog number H9658), NP polyclonal antibody and β-actin monoclonal antibody.

[0184] The results are as follows Figure 13-14 As shown, Fig.13 In the figure, A549 cells transfected with pRK5-HA empty vector are denoted as EV, and A549 cells overexpressing BAG6 protein are denoted as BAG6-HA; Fig.14 In the figure, + represents A549 cells overexpressing BAG6 protein, and - represents A549 cells transfected with pRK5-HA empty vector. It can be seen that compared with A549 cells transfected with pRK5-HA empty vector, overexpression of BAG6 protein significantly reduced the expression level of viral NP protein in A549 cells.

Claims

1. Use of a substance for inhibiting the expression of BAG6 gene or its encoded protein in any of the following: 1) Preparation and expansion of influenza virus products; 2) Preparation of products for promoting influenza virus replication; 3) Preparation of products for increasing the expression of influenza virus-related proteins; 4) Preparation of influenza virus for production of inactivated vaccines.

2. The use according to claim 1, characterized in that: The influenza virus is influenza A virus.

3. The use according to claim 1 or 2, characterized in that: The substance that inhibits the expression of the BAG6 gene or its encoded protein includes a CRISPR / Cas9 system that interferes with the expression of the BAG6 gene or its encoded protein.

4. A recombinant cell, wherein the expression of the BAG6 gene in a host cell is suppressed to obtain a recombinant cell.

5. The recombinant cell according to claim 4, characterized in that: The recombinant cell is prepared according to a method comprising the following steps: introducing the CRISPR / Cas9 system described in claim 3 into the host cell to inhibit the expression of the BAG6 gene in the host cell.

6. Use of the recombinant cell according to claim 4 or 5 in any of the following: 1) Preparation and expansion of influenza virus products; 2) Preparation of products for promoting influenza virus replication; 3) Prepare products for increasing the expression of influenza virus-related proteins.

7. Use of the BAG6 gene or its encoded protein or biological materials related thereto or substances that increase the expression of the BAG6 gene or its encoded protein in any of the following: 1) Preparing products for inhibiting influenza virus replication; 2) Preparing products for inhibiting the expression of influenza virus-related proteins; 3) Prepare anti-influenza virus products.

8. The use according to claim 7, characterized in that: The biological material related to the BAG6 gene is an expression box, a recombinant vector or a recombinant virus or a recombinant cell containing the BAG6 gene.

9. The use according to claim 7 or 8, characterized in that: The influenza virus is influenza A virus.

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