Influenza A virus culture medium containing zinc ions and preparation method

Abstract

The invention relates to the technical field of biology, and aims to provide a zinc ion-containing influenza A virus culture medium and a preparation method thereof. The culture medium contains a serum-free cell culture medium, water, a PBS (Phosphate Buffer Solution), bovine serum albumin with the final concentration of 20g / L, TPCK-Trypsin with the final concentration of 2mu g / mL and zinc ions with the final concentration of 0.009-0.015 mmol / L. According to the invention, zinc ions which are originally considered to generate a virus inhibition effect under a relatively low concentration condition can be used for promoting virus replication after the use concentration range is adjusted; therefore, a brand-new culture medium which can be applied to research on common cell models and virus in-vitro culture models of the influenza A virus is provided, and a new solution is provided for optimizing a vaccine production process. The culture medium is simple in formula and convenient to prepare; all the raw material components are common consumables in laboratories, and the required liquid components can be prefabricated in advance and refrigerated for later use; and large-scale vaccine production can be supported.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a culture medium for in vitro culture of influenza A virus and a preparation method thereof. Background Art

[0002] Influenza A virus is one of the main pathogens causing respiratory infections. It is susceptible to humans, pigs, poultry and other animals, posing a serious threat to livestock and poultry farming and human health. The difficulty in preventing and treating influenza A virus lies in the fact that its genome is composed of 8 segmented single-stranded negative-strand RNAs. Different influenza viruses can easily exchange gene fragments and undergo genetic recombination after infecting the same organism. So far, all human influenza pandemics have been caused by genetic recombination between human, pig and avian influenza viruses.

[0003] Vaccination is currently the main means of preventing influenza A virus. Due to the continuous mutation and genetic recombination of influenza viruses, the influenza vaccine must change its formula every year to cope with the prevalent strains, so universal influenza vaccines have become an extremely important research and development hotspot. Currently, the commonly used vaccines are mainly whole virus inactivated vaccines, live attenuated vaccines and subunit vaccines. The production of whole virus inactivated vaccines and live attenuated vaccines depends on in vitro virus culture. For example, the Madin-Darby canine kidney (MDCK) cell line is currently widely used in the production of inactivated influenza vaccines.

[0004] As influenza vaccines face great public and market demands, improving the efficiency of in vitro culture of influenza A viruses will be of great significance to vaccine production in many aspects. It can not only reduce the cost of vaccine production, but also shorten the vaccine development cycle, and provide the public with better quality and more effective vaccine products. However, the current influenza A virus culture medium has defects such as low production capacity and insufficient culture medium components, which leads to problems such as being unable to meet the needs of efficient virus production, high vaccine production costs, and heavy workload for operators.

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

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide an influenza A virus culture medium containing zinc ions and a preparation method thereof.

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

[0008] Provided is an influenza A virus culture medium containing zinc ions. The culture medium contains serum-free cell culture medium, water and PBS buffer, bovine serum albumin with a final concentration of 20 g / L, TPCK-Trypsin with a final concentration of 2 μg / mL, and zinc ions with 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 culture medium, MEM culture medium or F-12 culture medium.

[0010] As a preferred embodiment of the present invention, the source of the zinc ions is zinc sulfate heptahydrate.

[0011] The present invention further provides a method for preparing the above-mentioned influenza A virus culture medium containing zinc ions, comprising the following steps:

[0012] (1) Weigh an appropriate amount of bovine serum albumin and add it to a serum-free cell culture medium, fully dissolve and mix evenly, and filter with a sterile filter;

[0013] (2) preparing TPCK-Trypsin reagent with PBS buffer, filtering with a sterile filter, adding to the mixed solution obtained in step (1), and mixing evenly;

[0014] (3) preparing a solution with zinc sulfate heptahydrate and ultrapure water, filtering the solution with a sterile filter; diluting the solution with a liquid serum-free cell culture medium, and adding the solution to the mixed solution obtained in step (2); mixing the solution evenly to obtain an influenza A virus culture medium containing zinc ions;

[0015] The dosage relationship of the materials in each step is controlled so that in the final influenza A virus culture medium, the final concentration of bovine serum albumin 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 serum-free cell culture medium in the form of a liquid product; or, it is prepared by adding a dry powder serum-free cell culture medium product into ultrapure water and fully dissolving it.

[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 the step (2), the concentration of the TPCK-Trypsin reagent is 200 μg / mL, and it is added to the mixed solution obtained in the step (1) at a volume ratio of 1:100; in the step (3), the concentration of the zinc sulfate heptahydrate aqueous solution is 1.5 mol / L, and it is diluted to 1.5 mmol / L with serum-free cell culture medium at a ratio of 1:1000, and then added to the mixed solution obtained in the 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] The pre-prepared TPCK-Trypsin can be stored in a -80°C ultra-low temperature refrigerator, and the pre-prepared zinc sulfate heptahydrate solution can be sealed and stored in a 4°C refrigerator.

[0021] Description of the invention principle:

[0022] 1. In the common understanding of the technology in this field, zinc ions may be added to the culture medium during the cell or plant cultivation process according to the actual situation. The main purpose of this practice is to provide the nutrients required for the cell development of the cultivated object during the growth process (such as the record in the document "Zinc supplementation increases protein titer of recombinant CHO cells"). In addition, it is generally believed that the presence of zinc ions will inhibit bacterial growth and help create a sterile growth environment for the cultivated object (such as the record in the document "Progress in Research on the Mechanism of Antibacterial Action of Metal Ions Iron and Zinc on Acinetobacter baumannii"). Based on the above purposes, when zinc ions are added to the culture medium, their concentration is usually controlled within the range of 3μmol / L.

[0023] On the other hand, there are also research results suggesting that zinc ions can inhibit the replication of various respiratory viruses in vivo by activating the body's immunity (such as the literature "Zinc in Human Health and Infectious Diseases"), and oral zinc supplements are usually used to enhance the body's immunity. However, in in vitro experiments, the effective concentration range of zinc ions for antiviral effects is usually 100μmol / L to 1200μmol / L (such as the literature "The Role of Zinc in Antiviral Immunity"), and the specific concentration varies depending on the type of virus and the type of zinc salt.

[0024] 2. Based on long-term in-depth research, the inventor team found that when the concentration of zinc ions added to the culture medium is in the range of a final concentration of 0.009 to 0.015 mmol / L, it can effectively promote the replication of influenza A virus in cells, and the level of toxin production can be significantly improved. According to a large number of comparative experiments and theoretical analysis, its mechanism of action is summarized as follows: zinc ions not only maintain cell membrane stability, but also provide metal ligands for viral nucleases, accelerating the entry of viral particles into cells. The polymerase activity was detected by a dual luciferase activity detection experiment and it was found that zinc ions can significantly enhance the activity of influenza A virus polymerase. Therefore, the innovative use of zinc ions in the present invention breaks through the conventional cognition of those skilled in the art.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention overcomes the technical prejudice in the field and uses zinc ions, which were originally thought to have a virus-inhibiting effect at a relatively low concentration, to promote virus replication after adjusting the concentration range. This provides a new culture medium that can be used in common cell models and virus in vitro culture models for studying influenza A viruses, and provides a new solution for optimizing vaccine production processes.

[0027] 2. The influenza A virus culture medium proposed by the present invention has a simple formula and is easy to prepare; all raw material components are common laboratory consumables, and the required liquid components can be pre-prepared and refrigerated for standby use, and can be prepared when needed, avoiding problems such as contamination and failure caused by long-term refrigeration or repeated freezing and thawing.

[0028] 3. The present invention can support large-scale vaccine production. The composition of the culture medium is very important for the in vitro culture of cells and viruses, and a large part of the cost of large-scale in vitro culture is also reflected in the culture medium. Only a trace amount of zinc ions can be added to increase the virus titer in the culture medium, reduce the production cost of influenza virus vaccines, and improve vaccine production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of zinc ions promoting influenza A virus replication in MDCK cells in MEM medium.

[0030] Figure 2 Schematic diagram of zinc ions promoting the toxin titer of influenza A virus in MDCK cells in MEM medium.

[0031] Figure 3 Schematic representation of zinc ions promoting influenza A virus replication in A549 cells in F-12 medium.

[0032] Figure 4 Schematic diagram of zinc ions promoting the toxin titer of influenza A virus in A549 cells in F-12 medium.

[0033] Figure 5 Schematic diagram of zinc ions promoting influenza A virus replication in MDCK cells in DMEM medium.

[0034] Figure 6 Schematic diagram of zinc ions in DMEM medium promoting the toxin titer of influenza A virus in MDCK cells.

[0035] Figure 7 Schematic diagram of experimental results on the concentration of zinc ions promoting the replication of influenza A virus in vitro.

[0036] Figure 8 Schematic diagram of detecting how zinc ions increase the viral vRNA content in cells in the early stages of infection through fluorescent quantitative PCR.

[0037] Fig. 9 Schematic diagram of detecting how zinc ions increase the content of viral non-structural protein NS1 in cells in the early stage of infection by immunoblotting.

[0038] Fig.10 Schematic representation of zinc ions enhancing influenza A virus polymerase activity in A549 cells.

[0039] Fig.11 Schematic diagram of zinc ion enhancement of influenza A virus polymerase activity in 293T cells. DETAILED DESCRIPTION

[0040] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent consumables companies unless otherwise specified. 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] 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.

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

[0043] Part I Experimental evaluation of the effect of zinc ions on promoting viral replication

[0044] Example 1 Experimental evaluation of the effect of zinc ions on promoting the replication of influenza A virus in MDCK cells

[0045] 1. Preparation of Influenza A Virus Medium Containing Zinc Ions

[0046] The culture medium used for MDCK cells is a serum-free cell culture medium MEM in liquid form, which does not contain zinc ions. A certain dose of zinc sulfate heptahydrate solution is added to the serum-free MEM culture medium to make the final concentration of zinc ions 0.015 mmol / L.

[0047] The specific preparation operation is as follows: weigh an appropriate amount of bovine serum albumin, fully dissolve and mix with serum-free cell culture medium MEM, and filter into a sterile container through a 0.22μm sterile filter in a biological safety cabinet. Pre-prepare TPCK-Trypsin with a concentration of 200μg / mL with PBS buffer and filter through a 0.22μm sterile filter, and add it to the mixed system at a volume ratio of 1:100. Pre-prepare a zinc-containing solution with a concentration of 1.5mol / L with ultrapure water and zinc sulfate heptahydrate and filter through a 0.22μm sterile filter, dilute it to 1.5mmol / L with serum-free cell culture medium MEM at 1:1000, and add it to the mixed system. Control the dosage relationship of each material so that the final influenza A virus culture medium contains serum-free cell culture medium MEM, water and PBS buffer, as well as bovine serum albumin with a final concentration of 20g / L, TPCK-Trypsin with a final concentration of 2μg / mL, and zinc ions with a final concentration of 0.015mmol / L.

[0048] 2. Infection of MDCK cells in medium containing influenza A virus containing zinc ions

[0049] Prepare MDCK cells to be inoculated with the virus. Count the cells in one well before inoculation and calculate the amount of virus to be used according to MOI = 0.01. Add the virus to the influenza A virus culture medium containing zinc ions and mix well. The inoculated cells need to be washed twice with sterile PBS in advance, and then inoculated with influenza A virus A / Puerto Rico / 8 / 1934 (H1N1), and the supernatant is collected 24 hours after inoculation. The virus culture medium without additional zinc ions is used as the control group, and the same infection experiment is carried out, and the supernatant is collected 24 hours after inoculation.

[0050] 3. Influenza A virus vRNA assay

[0051] In the supernatant obtained in the previous step, influenza A virus vRNA was extracted, reverse transcribed into cDNA, and the amount of vRNA was measured by fluorescent quantitative 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 the infected culture supernatant, 20μL of Proteinase K and 200μL of Carrier RNA working solution, cover the tube, vortex and oscillate for 15 seconds, incubate at 56℃ for 15 minutes; add 250μL of anhydrous ethanol, cover the tube and vortex and oscillate for 15 seconds, and place at room temperature for 5 minutes; carefully transfer all the solution and flocculent precipitate in the centrifuge tube to the RNase-Free adsorption column, centrifuge at 6000g for 1 minute, discard the waste liquid, and put the adsorption column back into the collection tube; add 500μL of buffer GD, centrifuge at 6000g for 1 minute, discard the waste liquid, and put the adsorption column back into the collection tube; add 600μL of rinse solution PW, let it stand for 2 minutes, centrifuge at 6000g for 1 minute, discard the waste liquid, and repeat the rinse once; add 500μL of anhydrous ethanol, centrifuge at 6000g for 1 minute; after discarding the waste liquid, centrifuge at 12000g for 2 minutes to make the adsorption film completely dry; put the adsorption column into a new RNase-Free centrifuge tube and leave it open for 1 minute to allow the organic solvent to evaporate; drop 20μL of RNase-Free ddH2O, let stand at room temperature for 2 minutes, then centrifuge at 12000g for 1 minute to obtain vRNA product

[0053] The steps for reversing the vRNA product are as follows: according to the instructions of the HiScript II 1st Strand cDNA Synthesis Kit, add 4×gDNA wiper Mix to 12μL vRNA product and mix well; react at 42°C for 2 minutes in a gene thermal cycler to remove gDNA in the system; then add 2μL 10×RT Mix, 2μL HiScript II Enzyme Mix and 2pmol gene-specific primers, mix well, react at 50°C for 15 minutes and 85°C for 2 minutes in a gene thermal cycler to obtain the corresponding cDNA.

[0054] The steps of quantitative PCR for vRNA determination are as follows: add cDNA, 2×ChamQUniversal SYBR qPCR Master Mix, primer F, primer R, RNase-free H 2 O, the volumes were 1.75 μL, 35.0 μL, 1.4 μL, 1.4 μL, and 30.45 μL, a total of 70 μL, and after mixing, 20 μL / tube was divided into 3 duplicate wells. Each experiment was repeated three times independently. The expression level of host gene mRNA was standardized with the internal reference 18S rRNA and 2-ΔΔCt Formula calculation, each replicate well 2 -ΔΔCt After calculating the average value, the 2 -ΔΔCt The mean values ​​were analyzed for significance using paired two-tailed Student's t test and plotted using Graphpad Prism 9 software. Figure 1 As shown, during the infection of MDCK cells with influenza A virus, the level of vRNA of influenza A virus in the culture medium supernatant increased significantly after using influenza A virus culture medium containing zinc ions, proving that the replication level of influenza A virus in MDCK cells increased in the influenza A virus culture medium containing zinc ions.

[0055] 4. Influenza A virus titer determination

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

[0057] TCID 50 The determination steps are as follows: MDCK cells were inoculated into a 96-well plate 16 hours in advance, and after the cells formed a monolayer, the MDCK cells were washed twice with PBS buffer. The supernatant (10 -1 -10 -8 ) as infection wells, and set up two rows of negative control wells. Add 100 μL of MEM infection medium to each well and put it back into the carbon dioxide incubator for later use. After infection, put the cells into a cell culture incubator dedicated for virus infection at 34°C and 5% CO 2 Culture for 5-7 days, observe cytopathic effects 1-2 times a day, and mark the wells where cytopathic effects occur; record the positive results in each row and calculate the TCID of the virus sample using the Reed-Muench two-way method 50 The experimental results are as follows Figure 2 As shown, during the infection of MDCK cells with influenza A virus, the titer level of influenza A virus in the culture medium supernatant increased significantly after using influenza A virus culture medium containing zinc ions, proving that the toxin production level of influenza A virus in MDCK cells increased in the influenza A virus culture medium containing zinc ions.

[0058] Example 2 Experimental evaluation of the effect of zinc ions on promoting the replication of influenza A virus in A549 cells

[0059] 1. Preparation of Influenza A Virus Medium Containing Zinc Ions

[0060] The culture medium used for A549 cells is serum-free cell culture medium F-12, which itself contains 0.0015 mmol / L of zinc ions. A certain dose of zinc sulfate heptahydrate solution is added to the serum-free F-12 culture medium to make the final concentration of zinc ions 0.009 mmol / L.

[0061] The specific preparation operation is as follows: weigh an appropriate amount of bovine serum albumin, fully dissolve and mix with serum-free cell culture medium F-12, and filter into a sterile container through a 0.22μm sterile filter in a biological safety cabinet. Pre-prepare TPCK-Trypsin with a concentration of 200μg / mL using PBS buffer and filter through a 0.22μm sterile filter, and add it to the mixed system at a volume ratio of 1:100. Pre-prepare a zinc-containing solution with a concentration of 1.5mol / L using ultrapure water and zinc sulfate heptahydrate and filter through a 0.22μm sterile filter, dilute it to 1.5mmol / L with serum-free cell culture medium F-12 at a ratio of 1:1000, and add it to the mixed system. The dosage relationship of each material is controlled so that the final influenza A virus culture medium contains serum-free cell culture medium F-12, water and PBS buffer, as well as bovine serum albumin with a final concentration of 20 g / L, TPCK-Trypsin with a final concentration of 2 μg / mL, and zinc ions with a final concentration of 0.009 mmol / L.

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

[0063] Prepare A549 cells to be inoculated with the virus. Count the cells in one well before inoculation and calculate the amount of virus to be used at MOI = 0.01. Add the virus to the influenza A virus culture medium containing zinc ions and mix well. The inoculated cells need to be washed twice with sterile PBS in advance, and then inoculated with influenza A virus A / Puerto Rico / 8 / 1934 (H1N1), and the supernatant is collected 24 hours after inoculation. The virus culture medium without additional zinc ions is used as the control group, and the same infection experiment is carried out, and the supernatant is collected 24 hours after inoculation.

[0064] 3. Influenza A virus vRNA assay

[0065] The influenza A virus vRNA was extracted from the supernatant obtained in the previous step, and the amount of vRNA was measured by fluorescence quantitative PCR after reverse transcription into cDNA. Figure 3 As shown, during the infection of A549 cells with influenza A virus, after using influenza A virus culture medium containing zinc ions, the vRNA level of influenza A virus in the culture medium supernatant increased, proving that the replication level of influenza A virus in A549 cells increased in the influenza A virus culture medium containing zinc ions.

[0066] The influenza A virus vRNA was determined as in Example 1.

[0067] 4. Influenza A virus titer determination

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

[0069] TCID 50 The determination steps are 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 level of influenza A virus in the culture medium supernatant increased significantly after using influenza A virus culture medium containing zinc ions, proving that the toxin production level of influenza A virus in A549 cells increased in the influenza A virus culture medium containing zinc ions.

[0070] Example 3 Experimental evaluation of the effect of zinc ions on promoting the replication of influenza A virus in MDCK cells

[0071] 1. Preparation of Influenza A Virus Medium Containing Zinc Ions

[0072] The culture medium used for MDCK cells in this embodiment is a dry powdered serum-free cell culture medium DMEM, which does not contain zinc ions and is prepared into a liquid culture medium in advance with ultrapure water before use. A certain dose of zinc sulfate heptahydrate solution is added to the serum-free DMEM culture medium to make the final concentration of zinc ions 0.012 mmol / L.

[0073] The specific preparation operation is as follows: weigh an appropriate amount of bovine serum albumin, fully dissolve and mix with serum-free cell culture medium DMEM, and filter into a sterile container through a 0.22μm sterile filter in a biological safety cabinet. Pre-prepare TPCK-Trypsin with a concentration of 200μg / mL using PBS buffer and filter through a 0.22μm sterile filter, and add it to the mixed system at a volume ratio of 1:100. Pre-prepare a zinc-containing solution with a concentration of 1.5mol / L using ultrapure water and zinc sulfate heptahydrate and filter through a 0.22μm sterile filter, dilute it to 1.5mmol / L with serum-free cell culture medium DMEM at a ratio of 1:1000, and add it to the mixed system. The dosage relationship of each material is controlled so that the final influenza A virus culture medium contains serum-free cell culture medium DMEM, water and PBS buffer, as well as bovine serum albumin with a final concentration of 20 g / L, TPCK-Trypsin with a final concentration of 2 μg / mL, and zinc ions with a final concentration of 0.012 mmol / L.

[0074] 2. Infection of MDCK cells in medium containing influenza A virus containing zinc ions

[0075] Prepare MDCK cells to be inoculated with the virus. Count the cells in one well before inoculation and calculate the amount of virus to be used according to MOI = 0.01. Add the virus to the influenza A virus culture medium containing zinc ions and mix well. The inoculated cells need to be washed twice with sterile PBS in advance, and then inoculated with influenza A virus A / Puerto Rico / 8 / 1934 (H1N1), and the supernatant is collected 24 hours after inoculation. The virus culture medium without additional zinc ions is used as the control group, and the same infection experiment is carried out, and the supernatant is collected 24 hours after inoculation.

[0076] 3. Influenza A virus vRNA assay

[0077] The influenza A virus vRNA was extracted from the supernatant obtained in the previous step, and the amount of vRNA was measured by fluorescence quantitative PCR after reverse transcription into cDNA. Figure 5 As shown, during the infection of MDCK cells with influenza A virus, after using influenza A virus culture medium containing zinc ions, the vRNA level of influenza A virus in the culture supernatant increased, proving that the replication level of influenza A virus in A549 cells increased in the influenza A virus culture medium containing zinc ions.

[0078] The influenza A virus vRNA was determined as in Example 1.

[0079] 4. Influenza A virus titer determination

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

[0081] TCID 50 The determination steps are 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 level of influenza A virus in the culture medium supernatant increased significantly after using influenza A virus culture medium containing zinc ions, proving that the toxin production level of influenza A virus in A549 cells increased in the influenza A virus culture medium containing zinc ions.

[0082] Part II: Experimental verification of the mechanism of action of zinc ions

[0083] 1. First, in order to explore 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 method includes: infecting MDCK cells with H1N1 PR8 at an MOI of 1, adding zinc sulfate heptahydrate to the MEM culture medium to make the final concentration of zinc ions 0.006mmol / L, 0.009mmol / L, 0.012mmol / L, 0.015mmol / L, 0.018mmol / L, and 0.021mmol / L, and using the MEM culture medium without zinc sulfate heptahydrate as the control group; collecting samples 24 hours after infection for influenza A virus vRNA determination. The influenza A virus infection steps and vRNA determination method are the same as in Example 1. Figure 7 As shown, when the zinc ion concentration in the culture medium was in the range of 0.009 to 0.015 mmol / L, the replication of influenza A virus in MDCK cells was significantly enhanced.

[0085] 2. Validation experiment of zinc ions promoting influenza A virus replication in cells

[0086] To explore 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, and zinc sulfate heptahydrate was added to the F-12 culture medium to make the final concentration of zinc ions 0.009 mmol / L. The F-12 culture medium without additional zinc sulfate heptahydrate was used as the control group; after infection, the cells were placed at 4°C, and after 3 hours, the cells were rinsed 2 to 3 times with sterile cold PBS, and the cell samples were collected for immunoblotting experiments. The amount of virus that entered the cells was displayed by the content of viral NS1 protein. Figure 8 As shown, zinc ions promote the entry of influenza A virus into cells.

[0087] MT1M knockout cell line was constructed by CRISPR / Cas9 system. MT1M protein is a metallothionein, and its main physiological function is to maintain intracellular zinc ion homeostasis. Fig. 9 and Fig.10 As shown, in the MT1M knockout strain, the intracellular zinc ion content was reduced and the replication of influenza A virus was inhibited. Figure 7 In the experimental results, supplementing zinc ions in the culture medium can also promote the entry of influenza A virus into the MT1M-deficient cell line in the early stage of infection, proving that zinc ions can promote the entry of influenza A virus into cells.

[0088] The immunoblotting assay was performed as follows:

[0089] Cell protein extraction: discard the culture medium in the plate, wash twice with 1mL sterile pre-cooled PBS; after discarding the waste liquid, add 1mL PBS and scrape the adherent cells in the well with a cell scraper, transfer the cell suspension to a clean EP tube after blowing several times, centrifuge at 300g for 5min, discard the supernatant waste liquid to obtain a cell pellet; add 200μL protein lysis buffer RIPA (with protease inhibitor PMSF added in advance) to the cell pellet, let it stand on ice for 10min, centrifuge at 12000g, 4℃ for 10min, transfer the supernatant to a new 1.5mL centrifuge tube (about 200μL), and the protein sample is in the supernatant; finally, add the corresponding amount of 5× dual-color Loading Buffer to the supernatant, boil it at 100℃ in a metal bath for 5-10min, and store it in a -80℃ refrigerator after cooling or directly perform protein immunoblotting experiments.

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

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

[0092] Transfer: After electrophoresis, remove the glass plate, pry open the gel plate and cut each target protein according to the size of the marker; cut the PVDF transfer membrane of the corresponding size according to the gel strip, soak the PVDF membrane in methanol for 30 seconds and then soak it in the transfer solution; after the transfer filter paper and sponge are soaked in the transfer solution, assemble the transfer device in the order of sponge, transfer filter paper, PVDF membrane, protein gel strip, transfer filter paper and sponge, and leave no bubbles between the gel strip, PVDF membrane and transfer filter paper; after the transfer device is assembled, put the corresponding red and black surfaces into the transfer electrode; according to the size of the target protein, transfer the membrane at a constant current of 200mA for the corresponding time, wherein the protein below 30kDa is transferred for 15-20min, and the protein 30-100kDa is transferred for 20-40min.

[0093] Incubation with primary antibody: After blocking, wash the membrane with TBST, immerse the membrane in TBST and wash on a horizontal shaker at 50 r / min at room temperature for 5 minutes, repeat 3 times; dilute the corresponding protein primary antibody to the working concentration with the primary antibody diluent according to the instructions, immerse the corresponding membranes respectively, and incubate overnight at 4°C at 20 r / min for 14 to 16 hours.

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

[0095] Exposure: Exposure solution was prepared at a volume ratio of 1:1 between A solution and B solution, and was used immediately after preparation. After the incubation with the secondary antibody, the membrane was washed with TBST at 50 r / min at room temperature for 5 min, the waste liquid was discarded and repeated 3 times; the membrane was placed on a clean sample plate, a layer of exposure solution was evenly dropped on the surface of the membrane, and the membrane was placed in an automatic fluorescent gel imaging analyzer to view the distribution of proteins on the membrane using the software CapturePro.

[0096] 3. Validation experiment of zinc ion enhancing the polymerase activity of influenza A virus

[0097] The polymerase activity of influenza A virus was detected in A549 cells using the dual luciferase system. The experimental method was as follows: cells were prepared one day in advance and 3 × 10 5 A549 cells were plated in 6-well plates. When the cells grew to about 70-80%, 500 ng of PB1, PB2, PA, NP reverse genetic plasmids of H1N1 PR8, 200 ng of pPoll-NS-luc plasmid inserted with viral NS fragment promoter and 50 ng of pRLTK plasmid expressing Renilla luciferase were transfected into the cells. After 4 hours, the culture medium was replaced with F-12 medium containing different concentrations of zinc ions. The final concentrations of zinc ions in the F-12 medium of each group were 0.0015 mmol / L, 0.009 mmol / L and 0.015 mmol / L, respectively.

[0098] 24 hours after cell transfection in each well, remove the culture medium in the 6-well plate wells, add 1 mL of PBS, scrape the cells in the wells with a cell scraper, collect them into a clean EP tube after pipetting, and centrifuge at 300g for 3 minutes; after discarding the waste liquid, add 300 μL of lysis solution to the cell pellet, pipette it with a pipette to fully lyse it, and take the supernatant into a new EP tube after centrifugation. According to the instructions of the luciferase activity detection kit, the activity of dual luciferase was detected by microplate reader: the activity of Renilla luciferase was used as an internal reference to correct the effect of transfection efficiency on the experimental group. The algorithm for polymerase activity is the ratio of the firefly luciferase activity value to the Renilla luciferase activity value. The experimental group was compared with the control group to analyze the differences and significance, and the GraphPadPrism graphics software was used for graphics analysis. All experiments were repeated at least three times. Fig.11As shown in the figure, zinc ion supplementation can enhance the activity of influenza A virus polymerase; as the zinc ion concentration in the culture medium increases, the influenza A virus polymerase activity in A549 cells becomes stronger. When the zinc ion concentration increases to 0.015mmol / L, the influenza A virus polymerase activity can be enhanced by 3 to 4 times.

Claims

1. An influenza A virus culture medium containing zinc ions, characterized in that: The culture medium contains serum-free cell culture medium, water and PBS buffer, as well as bovine serum albumin with a final concentration of 20 g / L, TPCK-Trypsin with a final concentration of 2 μg / mL, and zinc ions with a final concentration of 0.009 to 0.015 mmol / L.

2. The influenza A virus culture medium according to claim 1, characterized in that The serum-free cell culture medium is DMEM culture medium, MEM culture medium or F-12 culture medium.

3. The influenza A virus culture medium according to claim 1, characterized in that The source of the zinc ion is zinc sulfate heptahydrate.

4. A method for preparing a culture medium for influenza A virus containing zinc ions, characterized in that: The steps include: (1) Weigh an appropriate amount of bovine serum albumin and add it to a serum-free cell culture medium, fully dissolve and mix evenly, and filter with a sterile filter; (2) preparing TPCK-Trypsin reagent with PBS buffer, filtering with a sterile filter, adding to the mixed solution obtained in step (1), and mixing evenly; (3) preparing a solution with zinc sulfate heptahydrate and ultrapure water, filtering the solution with a sterile filter; diluting the solution with a liquid serum-free cell culture medium, and adding the solution to the mixed solution obtained in step (2); mixing the solution evenly to obtain an influenza A virus culture medium containing zinc ions; The dosage relationship of the materials in each step is controlled so that in the final influenza A virus culture medium, the final concentration of bovine serum albumin 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.

5. The method according to claim 4, characterized in that The liquid serum-free cell culture medium is a serum-free cell culture medium in the form of a liquid product; or, it is prepared by adding a dry powder serum-free cell culture medium product into ultrapure water and fully dissolving it.

6. The method according to claim 4, characterized in that The pore size of the sterile filter is 0.22 μm.

7. The method according to claim 4, characterized in that In the step (2), the concentration of the TPCK-Trypsin reagent is 200 μg / mL, and it is added to the mixed solution obtained in step (1) at a volume ratio of 1:100; in the step (3), the concentration of the zinc sulfate heptahydrate aqueous solution is 1.5 mol / L, and it is diluted to 1.5 mmol / L with serum-free cell culture medium at a ratio of 1:1000, and then added to the mixed solution obtained in step (2).

8. The method according to claim 4, characterized in that 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.

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