A medium for culturing influenza a virus containing zinc ions and a method for preparing the same

Abstract

The application relates to the technical field of biology, and aims to provide a zinc ion-containing influenza A virus culture medium and a preparation method. The culture medium contains a serum-free cell culture medium, water and PBS buffer, and 20 g / L of bovine serum albumin with a final concentration, 2 ug / mL of TPCK-Trypsin with a final concentration, and 0.009-0.015 mmol / L of zinc ions with a final concentration. The zinc ions which are originally considered to have a virus inhibiting effect under a lower concentration condition can be used to promote virus replication after the concentration range is adjusted; thus, a brand-new culture medium which can be applied to a common cell model and a virus in-vitro culture model for studying the influenza A virus is provided, and a new solution for optimizing a vaccine production process is provided. The culture medium formula is simple and convenient to prepare; all the raw material components are common consumables in laboratories, the liquid components required can be prepared in advance and stored in cold storage for standby, and the culture medium can support mass vaccine production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a culture medium and preparation method for in vitro culture of influenza A virus. Background Technology

[0002] Influenza A virus is one of the main pathogens causing respiratory infections. Humans, pigs, and poultry are all susceptible, posing a serious threat to livestock farming and human health. The difficulty in controlling influenza A virus lies in the fact that its genome consists of eight segmented single-stranded negative-sense RNA molecules. Different influenza viruses can easily exchange gene segments after infecting the same organism, resulting in gene recombination. To date, all human influenza pandemics have been caused by gene recombination between human, swine, and avian influenza viruses.

[0003] Vaccination is currently the primary means of preventing influenza A virus infection. Because influenza viruses constantly mutate and recombine, influenza vaccines must be redesigned annually to address circulating strains, making universal influenza vaccines a crucial area of ​​research and development. Currently, commonly used vaccines include whole-virus inactivated vaccines, live attenuated vaccines, and subunit vaccines. The production of whole-virus inactivated vaccines and live attenuated vaccines relies on in vitro virus culture. For example, the Madin-Darby canine kidney (MDCK) cell line is widely used in the production of inactivated influenza vaccines.

[0004] Given the significant public and market demand for influenza vaccines, improving the efficiency of in vitro culture of influenza A viruses is of great importance to vaccine production in many ways. It can not only reduce vaccine production costs but also shorten the vaccine development cycle, providing the public with higher-quality and more effective vaccine products. However, current influenza A virus culture media suffer from low production capacity and insufficient culture medium components, leading to problems such as inability to meet the needs of efficient virus production, high vaccine production costs, and heavy workloads for operators.

[0005] Therefore, if a low-cost and easy-to-prepare in vitro culture medium for influenza A virus can be provided, and used to study common cell models and in vitro culture models of influenza A virus, it will play a very important role in optimizing vaccine production processes. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a zinc ion-containing culture medium for influenza A virus and a preparation method thereof.

[0007] To solve the technical problem, the solution of the present invention is:

[0008] A zinc-containing influenza A virus culture medium is provided, which contains serum-free cell culture medium, water and PBS buffer, as well as bovine serum albumin at a final concentration of 20 g / L, TPCK-Trypsin at a final concentration of 2 μg / mL, and zinc ions at a final concentration of 0.009–0.015 mmol / L.

[0009] As a preferred embodiment of the present invention, the serum-free cell culture medium is DMEM medium, MEM medium or F-12 medium.

[0010] As a preferred embodiment of the present invention, the zinc ions are sourced from zinc sulfate heptahydrate.

[0011] This invention further provides a method for preparing the aforementioned zinc ion-containing influenza A virus culture medium, comprising the following steps:

[0012] (1) Weigh an appropriate amount of bovine serum albumin and add it to a liquid serum-free cell culture medium. Dissolve and mix thoroughly, and then filter with a sterile filter.

[0013] (2) Prepare TPCK-Trypsin reagent with PBS buffer, filter it with a sterile filter, add it to the mixture obtained in step (1), and mix well.

[0014] (3) Prepare a solution with zinc sulfate heptahydrate and ultrapure water, and filter it with a sterile filter; dilute it with liquid serum-free cell culture medium and add it to the mixture obtained in step (2); mix well to obtain a zinc ion-containing influenza A virus culture medium;

[0015] Control the amount of materials used in each step so that the final concentration of bovine serum albumin in the influenza A virus culture medium is 20 g / L, the final concentration of TPCK-Trypsin is 2 μg / mL, and the final concentration of zinc ions is 0.009–0.015 mmol / L.

[0016] As a preferred embodiment of the present invention, the liquid serum-free cell culture medium is a product in liquid form; or, it is prepared by adding a dry powder serum-free cell culture medium product to ultrapure water and dissolving it thoroughly.

[0017] As a preferred embodiment of the present invention, the pore size of the sterile filter is 0.22 μm.

[0018] As a preferred embodiment of the present invention, in step (2), the concentration of TPCK-Trypsin reagent is 200 μg / mL, and it is added to the mixture obtained in step (1) at a volume ratio of 1:100; in step (3), the concentration of zinc sulfate heptahydrate aqueous solution is 1.5 mol / L, which is diluted to 1.5 mmol / L with serum-free cell culture medium at a ratio of 1:1000, and then added to the mixture obtained in step (2).

[0019] As a preferred embodiment of the present invention, the PBS buffer is prepared from sodium chloride, potassium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate and ultrapure water, wherein the concentrations of sodium chloride, potassium chloride, disodium hydrogen phosphate and dipotassium hydrogen phosphate are 8.0 g / L, 0.2 g / L, 14.4 g / L and 2.4 g / L, respectively.

[0020] Pre-prepared TPCK-Trypsin can be stored in an ultra-low temperature freezer at -80°C, and pre-prepared zinc sulfate heptahydrate solution can be sealed and stored in a freezer at 4°C.

[0021] Description of the invention principle:

[0022] 1. In the common understanding of the art, zinc ions may be added to the culture medium during cell or plant culture, depending on the specific circumstances. The main purpose of this practice is to provide the nutrients required for cell development during the growth process (as described in the literature "Zinc supplementation increases protein titer of recombinant CHO cells"). Furthermore, the presence of zinc ions is generally considered to inhibit bacterial growth, helping to create a sterile growth environment for the cultured organisms (as described in the literature "Research progress on the antibacterial mechanism of metal ions iron and zinc against Acinetobacter baumannii"). Based on these purposes, the concentration of zinc ions added to the culture medium is usually controlled within the range of 3 μmol / L.

[0023] On the other hand, some research suggests that zinc ions can inhibit the replication of various respiratory viruses in vivo by activating the body's immunity (e.g., the literature "Zinc in Human Health and Infectious Diseases"), usually through oral zinc supplementation to enhance the body's immunity. However, in in vitro experiments, the effective concentration range of zinc ions for antiviral effects is generally 100 μmol / L to 1200 μmol / L (e.g., the literature "The Role of Zinc in Antiviral Immunity"), with the specific concentration varying depending on the type of virus and the type of zinc salt.

[0024] 2. Based on long-term in-depth research, the inventors' team discovered that when the concentration of zinc ions added to the culture medium is within the final range of 0.009–0.015 mmol / L, it can effectively promote the replication of influenza A virus in cells, and significantly increase the level of viral production. According to numerous comparative experiments and theoretical analysis, the mechanism of action is summarized as follows: zinc ions both maintain cell membrane stability and provide metal ligands for viral nucleases, accelerating viral particle entry into cells. Polymerase activity assays using dual-luciferase activity detection revealed that zinc ions can significantly enhance the activity of influenza A virus polymerase. Therefore, the innovative use of zinc ions in this invention breaks through the conventional understanding of those skilled in the art.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. This invention overcomes the technical biases in the field by demonstrating that zinc ions, which were originally thought to have an inhibitory effect on viruses under low concentration conditions, can be used to promote viral replication after adjusting the concentration range. This provides a novel culture medium applicable to commonly used cell models and in vitro viral culture models for studying influenza A virus, offering a new solution for optimizing vaccine production processes.

[0027] 2. The influenza A virus culture medium formula proposed in this invention is simple and easy to prepare; all raw material components are common laboratory consumables, and the required liquid components can be pre-prepared and refrigerated for later use. It can be prepared as needed, avoiding problems such as contamination and failure caused by long-term refrigeration or repeated freeze-thaw cycles.

[0028] 3. This invention supports large-scale vaccine production. The composition of the culture medium is crucial for cell and virus in vitro culture, and a significant portion of the cost of large-scale in vitro culture is reflected in the culture medium. Adding only trace amounts of zinc ions can increase the virus titer in the culture medium, reduce the production cost of influenza virus vaccines, and improve vaccine production efficiency. Attached Figure Description

[0029] Figure 1 A schematic diagram illustrating how zinc ions promote the replication of influenza A virus in MDCK cells in MEM medium.

[0030] Figure 2 A schematic diagram illustrating how zinc ions promote the production of influenza A virus titers in MDCK cells in MEM medium.

[0031] Figure 3 A schematic diagram illustrating how zinc ions promote the replication of influenza A virus in A549 cells in F-12 medium.

[0032] Figure 4 A schematic diagram illustrating how zinc ions promote the production of influenza A virus titers in A549 cells in F-12 medium.

[0033] Figure 5 A schematic diagram illustrating how zinc ions promote the replication of influenza A virus in MDCK cells in DMEM medium.

[0034] Figure 6 A schematic diagram illustrating how zinc ions promote the production of influenza A virus titers in MDCK cells in DMEM medium.

[0035] Figure 7 A schematic diagram showing the experimental results of zinc ion concentration promoting the in vitro replication of influenza A virus.

[0036] Figure 8 A schematic diagram illustrating how zinc ions increase the level of viral vRNA in cells during the early stages of infection, as detected by quantitative real-time PCR.

[0037] Figure 9 A schematic diagram illustrating how the detection of zinc ions increases the content of the viral non-structural protein NS1 in cells during the early stages of infection.

[0038] Figure 10 A schematic diagram illustrating how zinc ions enhance the activity of influenza A virus polymerase in A549 cells.

[0039] Figure 11 A schematic diagram illustrating how zinc ions enhance the activity of influenza A virus polymerase in 293T cells. Detailed Implementation

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent and consumable companies. For example, TPCK-Trypsin powder was purchased from Sigma, and various types of serum-free cell culture medium powders or liquid culture media were purchased from Thermo Fisher Scientific.

[0041] The PBS buffer is prepared from sodium chloride, potassium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate and ultrapure water, with concentrations of sodium chloride, potassium chloride, disodium hydrogen phosphate and dipotassium hydrogen phosphate of 8.0 g / L, 0.2 g / L, 14.4 g / L and 2.4 g / L, respectively.

[0042] The influenza A virus strain A / Puerto Rico / 8 / 1934(H1N1) used for evaluation experiments in each embodiment was preserved by the College of Animal Sciences, Zhejiang University, and obtained through reverse genetic plasmid packaging.

[0043] Part 1: Evaluation Experiment of the Effect of Zinc Ions on Viral Replication

[0044] Example 1: Evaluation Experiment of the Effect of Zinc Ions on Promoting the Replication of Influenza A Virus in MDCK Cells

[0045] 1. Preparation of Zinc Ion-Containing Influenza A Virus Culture Medium

[0046] The culture medium used for MDCK cells was a serum-free liquid culture medium, MEM, which did not contain zinc ions. A certain amount of zinc sulfate heptahydrate solution was added to the serum-free MEM medium to achieve a final zinc ion concentration of 0.015 mmol / L.

[0047] The specific preparation steps are as follows: Weigh an appropriate amount of bovine serum albumin (BSA), dissolve and mix it thoroughly with serum-free cell culture medium MEM, and filter it through a 0.22 μm sterile filter into a sterile container in a biosafety cabinet. Prepare a 200 μg / mL TPCK-Trypsin solution using PBS buffer and filter it through a 0.22 μm sterile filter, then add it to the mixture at a volume ratio of 1:100. Prepare a 1.5 mol / L zinc-containing solution using ultrapure water and zinc sulfate heptahydrate and filter it through a 0.22 μm sterile filter. Dilute it to 1.5 mmol / L with serum-free cell culture medium MEM at a ratio of 1:1000 and add it to the mixture. Control the amounts of each material to ensure that the final influenza A virus culture medium contains serum-free cell culture medium MEM, water, PBS buffer, a final concentration of 20 g / L bovine serum albumin, a final concentration of 2 μg / mL TPCK-Trypsin, and a final concentration of 0.015 mmol / L zinc ions.

[0048] 2. Infecting MDCK cells with a zinc-containing influenza A virus culture medium.

[0049] Prepare MDCK cells for inoculation. Before inoculation, count the cells from one well and calculate the viral load using an MOI of 0.01. Add the virus to the influenza A virus culture medium containing zinc ions and mix well. Wash the cells twice with sterile PBS before inoculation, then inoculate with influenza A virus A / Puerto Rico / 8 / 1934 (H1N1). Collect the supernatant 24 hours after inoculation. Use virus culture medium without added zinc ions as a control group and perform the same infection experiment, collecting the supernatant 24 hours after inoculation.

[0050] 3. Influenza A virus vRNA assay

[0051] Influenza A virus vRNA was extracted from the supernatant obtained in the previous step, reverse transcribed into cDNA, and the amount of vRNA was determined by real-time PCR.

[0052] The steps for extracting influenza A virus vRNA are as follows: Extract vRNA from the sample: According to the instructions of the viral genomic DNA / RNA extraction kit, take an RNase / DNase-Free 1.5mL centrifuge tube, add 200μL of post-infection culture supernatant, 20μL of Proteinase K and 200μL of Carrier Oil. RNA working solution, cap the tube, vortex for 15 seconds, incubate at 56°C for 15 minutes; add 250 μL of anhydrous ethanol, cap the tube and vortex for 15 seconds, incubate at room temperature for 5 minutes; carefully transfer all solution and flocculent precipitate from the centrifuge tube to an RNase-Free adsorption column, centrifuge at 6000g for 1 minute, discard the waste liquid, and return the adsorption column to the collection tube; add 500 μL of buffer GD, centrifuge at 6000g for 1 minute, discard the waste liquid, and return the adsorption column to the collection tube; add 600 μL of wash buffer PW, let stand for 2 minutes, centrifuge at 6000g for 1 minute, discard the waste liquid, and repeat the wash once; add 500 μL of anhydrous ethanol, centrifuge at 6000g for 1 minute; discard the waste liquid, centrifuge at 12000g for 2 minutes to completely dry the adsorption membrane; place the adsorption column in a new RNase-Free centrifuge tube, and leave it open for 1 minute to allow the organic solvent to evaporate; add 20 μL of ethanol dropwise to the center of the adsorption membrane. RNase-free ddH2O was added and allowed to stand at room temperature for 2 minutes, then centrifuged at 12000g for 1 minute at room temperature to obtain the vRNA product.

[0053] The vRNA product reversal procedure is as follows: According to the HiScript II 1st Strand cDNA Synthesis Kit instructions, add 4×gDNA wiper Mix to 12μL of vRNA product and mix well; incubate at 42℃ for 2min in a gene amplification instrument to remove gDNA from the system; then add 2μL 10×RT Mix, 2μL HiScript II Enzyme Mix and 2pmol gene-specific primers, mix well, and incubate at 50℃ for 15min and 85℃ for 2min in a gene amplification instrument to obtain the corresponding cDNA.

[0054] The steps for determining vRNA using quantitative real-time PCR are as follows: Add cDNA, 2×ChamQ Universal SYBR qPCR Master Mix, primer F, primer R, and RNase-free H2O sequentially to the PCR reaction tube in volumes of 1.75 μL, 35.0 μL, 1.4 μL, 1.4 μL, and 30.45 μL, respectively, for a total of 70 μL. Mix well and aliquot into three wells (20 μL / tube). Each experiment is independently repeated three times. The expression level of host gene mRNA is standardized using an internal control 18S rRNA and analyzed using 2... -ΔΔCtFormula calculation, each complex hole 2 -ΔΔCt After calculating the average, the 2 of each independent repeated experiment -ΔΔCt The mean values ​​were analyzed for significance using a paired two-tailed Student's t-test, and graphs were plotted using Graphpad Prism 9 software. The experimental results are as follows: Figure 1 As shown, during the infection of MDCK cells with influenza A virus, the level of influenza A virus vRNA in the supernatant of the culture medium containing zinc ions was significantly increased after using influenza A virus culture medium containing zinc ions, which proves that the replication level of influenza A virus in MDCK cells is increased.

[0055] 4. Influenza A virus titer determination

[0056] Determine the TCID of the supernatant obtained in step 2. 50 Virus titer analysis was performed.

[0057] TCID 50 The assay steps are as follows: MDCK cells are seeded into 96-well plates 16 hours in advance. After the cells form a monolayer, the MDCK cells are washed twice with PBS buffer. The supernatant collected in step 2 (10... -1 -10 -8 Two rows of negative control wells were set up as infection wells. 100 μL of MEM infection medium was added to each well, and the cells were returned to the CO2 incubator for later use. After inoculation, the cells were placed in a virus-infected cell culture incubator and cultured at 34°C and 5% CO2 for 5-7 days. Cytopathic effects were observed 1-2 times daily, and wells showing cytopathic effects were marked. The TCID of the virus sample was calculated using the Reed-Muench two-factor method after recording the positive results for each row. 50 The experimental results are as follows: Figure 2 As shown, during the infection of MDCK cells with influenza A virus, the titer of influenza A virus in the supernatant of the culture medium containing zinc ions was significantly increased after using the influenza A virus culture medium containing zinc ions, which proves that the production level of influenza A virus in MDCK cells is increased.

[0058] Example 2: Evaluation Experiment of the Effect of Zinc Ions on Promoting the Replication of Influenza A Virus in A549 Cells

[0059] 1. Preparation of Zinc Ion-Containing Influenza A Virus Culture Medium

[0060] The culture medium used for A549 cells was serum-free cell culture medium F-12, which itself contains 0.0015 mmol / L of zinc ions. By adding a certain amount of zinc sulfate heptahydrate solution to the serum-free F-12 medium, the final concentration of zinc ions was made up to 0.009 mmol / L.

[0061] The specific preparation steps are as follows: Weigh an appropriate amount of bovine serum albumin, dissolve and mix it thoroughly with serum-free cell culture medium F-12, and filter it into a sterile container through a 0.22 μm sterile filter in a biosafety cabinet. Pre-prepare a 200 μg / mL TPCK-Trypsin solution with PBS buffer and filter it through a 0.22 μm sterile filter, then add it to the mixture at a volume ratio of 1:100. Pre-prepare a 1.5 mol / L zinc-containing solution with ultrapure water and zinc sulfate heptahydrate and filter it through a 0.22 μm sterile filter, then dilute it to 1.5 mmol / L with serum-free cell culture medium F-12 at a ratio of 1:1000 and add it to the mixture. The amounts of each material were controlled to ensure that the final influenza A virus culture medium contained serum-free cell culture medium F-12, water and PBS buffer, as well as bovine serum albumin at a final concentration of 20 g / L, TPCK-Trypsin at a final concentration of 2 μg / mL, and zinc ions at a final concentration of 0.009 mmol / L.

[0062] 2. Infection of A549 cells with influenza A virus culture medium containing zinc ions.

[0063] Prepare A549 cells for inoculation. Before inoculation, count the cells from one well and calculate the viral load using an MOI of 0.01. Add the virus to the influenza A virus culture medium containing zinc ions and mix well. Wash the cells twice with sterile PBS before inoculation, then inoculate with influenza A virus A / Puerto Rico / 8 / 1934 (H1N1). Collect the supernatant 24 hours after inoculation. Use virus culture medium without added zinc ions as a control group and perform the same infection experiment, collecting the supernatant 24 hours after inoculation.

[0064] 3. Influenza A virus vRNA assay

[0065] Influenza A virus vRNA was extracted from the supernatant obtained in the previous step, reverse transcribed into cDNA, and the amount of vRNA was determined by real-time PCR. The experimental results are as follows: Figure 3 As shown, during the infection of A549 cells with influenza A virus, the level of influenza A virus vRNA in the supernatant increased after using influenza A virus culture medium containing zinc ions, demonstrating that the replication level of influenza A virus in A549 cells is increased in influenza A virus culture medium containing zinc ions.

[0066] The assay for influenza A virus vRNA was the same as in Example 1.

[0067] 4. Influenza A virus titer determination

[0068] Determine the TCID of the supernatant obtained in step 2. 50Virus titer analysis was performed.

[0069] TCID 50 The measurement procedure is the same as in Example 1. The experimental results are as follows: Figure 4 As shown, during the infection of A549 cells with influenza A virus, the titer of influenza A virus in the supernatant of the culture medium containing zinc ions was significantly increased after using the influenza A virus culture medium containing zinc ions, which proves that the production level of influenza A virus in A549 cells is increased in the influenza A virus culture medium containing zinc ions.

[0070] Example 3: Evaluation Experiment of the Effect of Zinc Ions on Promoting the Replication of Influenza A Virus in MDCK Cells

[0071] 1. Preparation of Zinc Ion-Containing Influenza A Virus Culture Medium

[0072] In this embodiment, the culture medium used for MDCK cells was DMEM, a dry powder serum-free cell culture medium. This medium itself does not contain zinc ions and was pre-prepared into a liquid state with ultrapure water before use. A certain amount of zinc sulfate heptahydrate solution was added to the serum-free DMEM medium to achieve a final zinc ion concentration of 0.012 mmol / L.

[0073] The specific preparation steps are as follows: Weigh an appropriate amount of bovine serum albumin, thoroughly dissolve and mix it with serum-free cell culture medium DMEM, and filter it into a sterile container through a 0.22 μm sterile filter in a biosafety cabinet. Pre-prepare a 200 μg / mL TPCK-Trypsin solution using PBS buffer and filter it through a 0.22 μm sterile filter, then add it to the mixture at a volume ratio of 1:100. Pre-prepare a 1.5 mol / L zinc-containing solution using ultrapure water and zinc sulfate heptahydrate and filter it through a 0.22 μm sterile filter, then dilute it to 1.5 mmol / L with serum-free cell culture medium DMEM at a ratio of 1:1000 and add it to the mixture. The amounts of each material were controlled to ensure that the final influenza A virus culture medium contained serum-free cell culture medium DMEM, water, PBS buffer, bovine serum albumin at a final concentration of 20 g / L, TPCK-Trypsin at a final concentration of 2 μg / mL, and zinc ions at a final concentration of 0.012 mmol / L.

[0074] 2. Infecting MDCK cells with a zinc-containing influenza A virus culture medium.

[0075] Prepare MDCK cells for inoculation. Before inoculation, count the cells from one well and calculate the viral load using an MOI of 0.01. Add the virus to the influenza A virus culture medium containing zinc ions and mix well. Wash the cells twice with sterile PBS before inoculation, then inoculate with influenza A virus A / Puerto Rico / 8 / 1934 (H1N1). Collect the supernatant 24 hours after inoculation. Use virus culture medium without added zinc ions as a control group and perform the same infection experiment, collecting the supernatant 24 hours after inoculation.

[0076] 3. Influenza A virus vRNA assay

[0077] Influenza A virus vRNA was extracted from the supernatant obtained in the previous step, reverse transcribed into cDNA, and the amount of vRNA was determined by real-time PCR. The experimental results are as follows: Figure 5 As shown, during the infection of MDCK cells with influenza A virus, the level of influenza A virus vRNA in the supernatant of the culture medium increased after using influenza A virus culture medium containing zinc ions, proving that the replication level of influenza A virus in A549 cells is increased in the influenza A virus culture medium containing zinc ions.

[0078] The assay for influenza A virus vRNA was the same as in Example 1.

[0079] 4. Influenza A virus titer determination

[0080] Determine the TCID of the supernatant obtained in step 2. 50 Virus titer analysis was performed.

[0081] TCID 50 The measurement procedure is the same as in Example 1. The experimental results are as follows: Figure 6 As shown, during the infection of A549 cells with influenza A virus, the titer of influenza A virus in the supernatant of the culture medium containing zinc ions was significantly increased after using the influenza A virus culture medium containing zinc ions, which proves that the production level of influenza A virus in A549 cells is increased in the influenza A virus culture medium containing zinc ions.

[0082] Part Two: Experiments to Verify the Mechanism of Action of Zinc Ions

[0083] 1. First, in order to investigate the optimal concentration of zinc ions to promote the in vitro replication of influenza A virus, the applicant conducted experiments on MDCK cells.

[0084] Specifically, the procedure included: infecting MDCK cells with H1N1 PR8 at an MOI of 1; adding zinc sulfate heptahydrate to MEM medium to achieve final zinc ion concentrations of 0.006 mmol / L, 0.009 mmol / L, 0.012 mmol / L, 0.015 mmol / L, 0.018 mmol / L, and 0.021 mmol / L, with MEM medium without added zinc sulfate heptahydrate serving as a control; and collecting samples 24 hours after infection for influenza A virus vRNA assay. The influenza A virus infection procedure and vRNA assay method were the same as in Example 1. Figure 7 As shown, when the zinc ion concentration in the culture medium is in the range of 0.009–0.015 mmol / L, the replication of influenza A virus in MDCK cells is significantly enhanced.

[0085] 2. Verification experiment on the effect of zinc ions on the replication of influenza A virus in cells.

[0086] To investigate whether zinc ions affect the entry of influenza A virus into cells, the applicant conducted experiments on A549 cells. A549 control cells and MT1M protein knockout cell lines were infected with H1N1 PR8 at an MOI of 1. Zinc sulfate heptahydrate was added to F-12 medium to achieve a final zinc ion concentration of 0.009 mmol / L, with F-12 medium without additional zinc sulfate heptahydrate serving as the control group. After inoculation, cells were placed at 4°C, and after 3 hours, they were washed 2–3 times with sterile cold PBS. Cell samples were collected and subjected to Western blotting experiments. The viral load was indicated by the concentration of the viral NS1 protein. Figure 8 As shown, zinc ions promote the entry of influenza A virus into cells.

[0087] MT1M knockout cell lines were constructed using the CRISPR / Cas9 system. MT1M protein is a metallosulfonic acid protease whose main physiological function is to maintain intracellular zinc ion homeostasis. Figure 9 and Figure 10 As shown, intracellular zinc ion levels were reduced in the MT1M knockout strain, and influenza A virus replication was inhibited. Figure 7 The experimental results showed that zinc ions supplementing the culture medium could also promote the entry of early-stage influenza A virus into MT1M-deficient cell lines, proving that zinc ions can promote the entry of influenza A virus into cells.

[0088] The immunoblotting assay method is as follows:

[0089] Cell protein extraction: Discard the culture medium in the plate and wash twice with 1 mL of sterile pre-cooled PBS; after discarding the waste liquid, add 1 mL of PBS and scrape adherent cells from the wells with a cell scraper. After several pipetting cycles, transfer the cell suspension to a clean EP tube and centrifuge at 300 g for 5 min. Discard the supernatant to obtain the cell pellet; add 200 μL of protein lysis buffer RIPA (pre-added with protease inhibitor PMSF) to the cell pellet, incubate on ice for 10 min, then centrifuge at 12000 g at 4 °C for 10 min. Transfer the supernatant to a new 1.5 mL centrifuge tube (approximately 200 μL), with the protein sample in the supernatant; finally, add the corresponding amount of 5× dual-color loading buffer to the supernatant, boil in a metal bath at 100 °C for 5–10 min, cool, and store at -80 °C or directly perform Western blotting experiments.

[0090] Prepare SDS-PAGE protein electrophoresis gel: Wash the glass plates for gel preparation with running tap water, assemble them, and clamp them on the gel preparation rack to check for leaks; prepare the stacking gel and separating gel according to the instructions of the one-step gel preparation kit, pour the stacking gel into the space between the glass plates first, then pour the separating gel, insert a 15-well comb, and place at room temperature for 30-40 minutes until the gel solidifies.

[0091] Electrophoresis: After the protein electrophoresis gel solidifies, remove the glass gel plate from the gel casting rack, assemble the gel plate and electrodes, fill the inside of the glass plate with fresh electrophoresis buffer to check for leaks; load the processed protein sample and protein marker at 5-10 μL / well, add an appropriate amount of electrophoresis buffer to the outside of the glass plate, connect the electrodes, and perform electrophoresis at a constant voltage of 200V for 45 minutes.

[0092] Transfer: After electrophoresis, remove the glass plate, pry open the gel plate, and cut out the target proteins according to the marker size; cut out the corresponding size of PVDF transfer membrane according to the gel strip, soak the PVDF membrane in methanol for 30 seconds, and then soak it in the transfer buffer; after soaking the transfer filter paper and sponge in the transfer buffer, assemble the transfer device in the following order: sponge, transfer filter paper, PVDF membrane, protein gel strip, transfer filter paper, sponge, ensuring no air bubbles are left between the gel strip, PVDF membrane, and transfer filter paper; after assembling the transfer device, place it in the transfer electrode with the red and black sides facing each other; depending on the size of the target protein, transfer at a constant current of 200mA for the corresponding time, with proteins below 30kDa transferred in 15-20 min and proteins between 30-100kDa transferred in 20-40 min.

[0093] Primary antibody incubation: After blocking, wash the membrane with TBST, immerse the membrane in TBST and wash for 5 min at room temperature on a horizontal shaker at 50 r / min, repeat 3 times; dilute the corresponding protein primary antibody to the working concentration with primary antibody dilution buffer according to the instructions, immerse the corresponding membrane, and incubate overnight at 4℃ and 20 r / min for 14-16 h.

[0094] Secondary antibody incubation: Wash the membrane with TBST. Immerse the membrane in TBST on a horizontal shaker at 50 rpm for 5 min at room temperature, repeating 3 times. Dilute the secondary antibody to the working concentration according to the instructions, immerse the corresponding membranes, and incubate at 20 rpm for 2 h at room temperature.

[0095] Exposure: Prepare the exposure solution at a volume ratio of solution A to solution B of 1:1, and use immediately. After incubation with the secondary antibody, wash the membrane with TBST at 50 rpm at room temperature for 5 min, discard the waste solution, and repeat 3 times. Place the membrane on a clean sample plate, evenly drop a layer of exposure solution onto the membrane surface, and place it in an automated fluorescent gel imaging analyzer to view the protein distribution on the membrane using CapturePro software.

[0096] 3. Verification experiment on the enhancement of polymerase activity of influenza A virus by zinc ions

[0097] The polymerase activity of influenza A virus in A549 cells was detected using a dual-luciferase system. The experimental method is as follows: Prepare cells one day in advance, and press 3 × 10⁶ cells per well. 5 A549 cells were seeded into 6-well plates. When the cells reached a confluence of 70-80%, they were transfected with 500 ng each of the H1N1 PR8 reverse genetic plasmids PB1, PB2, PA, and NP, 200 ng of the pPoll-NS-luc plasmid containing the viral NS fragment promoter, and 50 ng of the pRLTK plasmid expressing René luciferase. Four hours later, the medium was replaced with F-12 medium containing different concentrations of zinc ions. The final zinc ion concentrations in the F-12 medium for each group were 0.0015 mmol / L, 0.009 mmol / L, and 0.015 mmol / L, respectively.

[0098] Twenty-four hours after cell transfection, the culture medium in each well of a 6-well plate was removed, 1 mL of PBS was added, and cells were scraped from the wells using a cell scraper. After pipetting, the cells were collected into clean EP tubes and centrifuged at 300g for 3 min. The waste liquid was discarded, and 300 μL of lysis buffer was added to the cell pellet. The cells were thoroughly lysed by pipetting, and the supernatant was collected into new EP tubes after centrifugation. The activity of dual-luciferase was measured using a microplate reader according to the instructions of the luciferase activity assay kit. Renida luciferase activity was used as an internal control to correct for the influence of transfection efficiency on the experimental groups. Polymerase activity was calculated as the ratio of firefly luciferase activity to Renida luciferase activity. Differences and significance were analyzed between the experimental and control groups, and graph analysis was performed using GraphPadPrism software. All experiments were repeated at least three times. Figure 11As shown, zinc ion supplementation can enhance the activity of influenza A virus polymerase; the higher the zinc ion concentration in the culture medium, the stronger the influenza A virus polymerase activity in A549 cells. When the zinc ion concentration increases to 0.015 mmol / L, the influenza A virus polymerase activity can be increased by 3 to 4 times.

Claims

1. A medium for culturing influenza A virus containing zinc ions, characterized in that, The medium contains serum-free cell culture medium, water and PBS buffer, and 20 g / L of bovine serum albumin, 2 μg / mL of TPCK-Trypsin, and 0.009-0.015 mmol / L of zinc ions; the serum-free cell culture medium is DMEM medium, MEM medium or F-12 medium; and the source of the zinc ions is zinc sulfate heptahydrate.

2. A method for preparing a culture medium for influenza A virus containing zinc ions, characterized by, The method comprises the following steps: (1) a proper amount of bovine serum albumin is added into liquid serum-free cell culture medium, and is dissolved and mixed uniformly, and is filtered by a sterile filter; (2) TPCK-Trypsin reagent is prepared by using PBS buffer, and is filtered by a sterile filter, and is added into the mixture obtained in step (1) and mixed uniformly; (3) zinc sulfate heptahydrate and ultrapure water are used to prepare a solution, which is filtered by a sterile filter, and is diluted by liquid serum-free cell culture medium, and is added into the mixture obtained in step (2), and is mixed uniformly to obtain the zinc ion-containing influenza A virus culture medium; The amount of each material in each step is controlled so that the final influenza A virus culture medium contains 20 g / L of bovine serum albumin, 2 μg / mL of TPCK-Trypsin, and 0.009-0.015 mmol / L of zinc ions.

3. The method of claim 2, wherein, The liquid serum-free cell culture medium is a product in the form of liquid serum-free cell culture medium, or is prepared by adding ultrapure water into a dry powder of serum-free cell culture medium.

4. The method of claim 2, wherein, The pore size of the sterile filter is 0.22 μm.

5. The method of claim 2, wherein, In step (2), the concentration of the TPCK-Trypsin reagent is 200 μg / mL, which is added into the mixture obtained in step (1) at a volume ratio of 1:100; and in step (3), the concentration of the zinc sulfate heptahydrate solution is 1.5 mol / L, which is diluted to 1.5 mmol / L by serum-free cell culture medium at a ratio of 1:1000, and then is added into the mixture obtained in step (2). The pore size of the sterile filter is 0.22 μm. In step (2), the concentration of the TPCK-Trypsin reagent is 200 μg / mL, which is added into the mixture obtained in step (1) at a volume ratio of 1:100; and in step (3), the concentration of the zinc sulfate heptahydrate solution is 1.5 mol / L, which is diluted to 1.5 mmol / L by serum-free cell culture medium at a ratio of 1:1000, and then is added into the mixture obtained in step (2).

Images