Method for efficiently amplifying RNA (Ribonucleic Acid) virus by adding xylinic acid and application

Abstract

The invention belongs to the technical field of cell engineering, and particularly discloses a method for efficiently amplifying RNA viruses by adding xylinic acid and application, and the method comprises the following steps: exogenously adding xylinic acid into a culture medium of eukaryotic cells, and infecting the eukaryotic cells by using the RNA viruses to promote efficient amplification of the RNA viruses. The invention provides an efficient amplification method for the fields of vaccine development based on the RNA virus, antiviral drug screening and the like, so that a basis is provided for producing, researching and developing anti-RNA virus vaccines or candidate drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering, and in particular relates to a method and application of adding lignoceric acid to efficiently amplify RNA viruses. Background Art

[0002] Viruses are pathogens composed of RNA or DNA and proteins. Diseases caused by viruses seriously endanger human health and life. Therefore, the understanding of the infection mechanism and biological characteristics of these malignant infectious viruses and the development of highly effective antiviral therapeutic drugs have become hot research topics in various countries. The premise of these studies is that we need to obtain enough viruses.

[0003] At present, the main methods for virus amplification are animal inoculation, chicken embryo inoculation and cell culture. The first two methods have some disadvantages. The animal inoculation method is affected by the body's immunity, and the virus cannot grow to the maximum. Many viruses lack specific infection indicators when cultured and amplified in chicken embryos. Relatively speaking, the cell culture method can avoid these disadvantages, and has the advantages of large inoculation volume, artificially controllable conditions, high vaccine quality and yield, and has become the most commonly used virus amplification method. Commonly used sensitive cell lines involved in the cell culture method include astroglioma cell line DBT-1, African green monkey kidney cell Vero and human lung cancer cell line A549, which can be used as platform cells for in vitro virus infection screening of drugs and vaccine development. In addition to the above advantages, virus amplification can also be intervened by cell line modification, for example, by knocking out antiviral related genes p53, IFITM3 and IFNAR1 to increase virus amplification. The cell culture method can also intervene in virus amplification by adding exogenous and exogenous additives. Studies have shown that folic acid metabolism negatively regulates 2'-5' oligoadenylate synthetase (OAS)-mediated antiviral natural immunity by inducing exogenous dsRNA accumulation; serine metabolism can also regulate the body's innate immunity to viral infection. Therefore, we can try to intervene in viral amplification by exogenously adding or endogenously reducing related substances.

[0004] When the virus infects cells, its nucleic acid molecules will bind to the pattern recognition receptors RLR, TLR or NLR in the host, further activating downstream transcription factors NF-κB and IRF3, etc. After these transcription factors enter the cell nucleus, they will promote the expression and release of interferon, and then interferon will cause a cascade reaction to neighboring cells or its own cells, thereby exerting an antiviral effect. Studies have shown that certain fatty acids can participate in the process of cellular immune regulation. Studies have found that during the infection of the DNA virus HSV-1 simple herpes virus, myristic acid can mediate the N-myristoylation modification of ARF1, an important regulatory factor of membrane transport STING, thereby promoting the reverse transport of STING to the Golgi apparatus and binding to LC3 to form autophagosomes, promoting STING-dependent autophagy and degradation. In addition, lipids are one of the important nutrients required by the human body and play an important role in the process of viral infection. For example, studies have shown that linoleic acid specifically binds to SARS-CoV-2 and can be used to target drugs that prevent this coronavirus from infecting human cells, which may help eliminate the disease. These reports suggest that screening potential functional lipid components will provide a method for intervening in viral amplification. Summary of the invention

[0005] In order to solve the above problems, the primary purpose of the present invention is to provide a method and application of adding lignoceric acid to efficiently amplify RNA viruses.

[0006] The specific technical solutions of the present invention include:

[0007] As a first aspect of the present invention, the present invention provides a method for efficiently amplifying RNA viruses by adding lignans, wherein lignans are exogenously added to the culture medium of eukaryotic cells, and then the eukaryotic cells are infected with RNA viruses to promote efficient amplification of RNA viruses.

[0008] As a further optimized solution of the present invention, the RNA virus is Newcastle disease virus NDV or vesicular stomatitis virus VSV.

[0009] As a further optimization scheme of the present invention, the added concentration of the lignoceric acid is 0.1-0.5 μM.

[0010] As a further optimization scheme of the present invention, the wood vinegar reduces the transcription levels of RIG-1, ISG15, IFIT1, IFIT2, MX1, TNFα and IFNβ in eukaryotic cells, thereby reducing the innate immune antiviral response in eukaryotic cells, thereby promoting intracellular viral infection and improving the replication ability of RNA viruses.

[0011] As a further optimization scheme of the present invention, the time for exogenous treatment of cells with wood acetate is 6-48 hours.

[0012] As a further optimized solution of the present invention, the eukaryotic cells are DBT-1 cells.

[0013] As a further optimization scheme of the present invention, the virus infection time during which the lignoceric acid promotes RNA virus replication is 16-20 hours.

[0014] As a second aspect of the present invention, the present invention also provides the use of any of the above-mentioned methods for efficiently amplifying RNA viruses by adding lignoceric acid in the production and development of anti-RNA virus vaccines or candidate drugs.

[0015] In summary, the beneficial effects of the present invention are:

[0016] The present invention affects virus infection by exogenously adding fatty acid-lignoceric acid into eukaryotic cells DBT-1. Lignoceric acid added to glioma cells DBT-1 within a certain range can significantly promote the amplification of RNA viruses. The present invention provides an efficient amplification method for the fields of RNA virus-based vaccine development and antiviral drug screening, thereby providing a basis for the production and development of anti-RNA virus vaccines or candidate drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of pyroligneous acid (C24:0) on the proliferation of DBT-1 cells;

[0018] Figure 2 Effects of exogenously added xyloic acid concentration gradient on the viral replication ability of NDV-GFP (A) and VSV (B);

[0019] Figure 3 Effects of exogenous addition of lignoceric acid in a time gradient on the viral replication capacity of NDV-GFP (A) and VSV (B);

[0020] Figure 4 Effects of exogenous addition of lignoceric acid on the transcription levels of NDV-GFP (A) and VSV-G (B);

[0021] Figure 5 This is the effect of lignoceric acid on the expression of cytokines and immune genes before and after RNA virus infection. DETAILED DESCRIPTION

[0022] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0023] 1. Materials and Reagents

[0024] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0025] Glioma cell DBT-1: donated by the Military Medical Research Institute of the Academy of Military Sciences or purchased from commercial sources.

[0026] All standard fatty acids were purchased from Sigma.

[0027] Newcastle disease virus NDV-GFP, vesicular stomatitis virus VSV: kindly donated by the Military Medical Research Institute of the Academy of Military Sciences.

[0028] The complete culture medium is DMEM culture medium containing 10% fetal bovine serum. Both the fetal bovine serum and the DMEM culture medium are purchased from commercial sources.

[0029] All primers were synthesized by Universal Company.

[0030] The quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0031] 2. Methods

[0032] The experimental methods in the following examples are all conventional biochemical methods unless otherwise specified.

[0033] 1. MTT assay to detect cell viability

[0034] When the experimental cell DBT-1 grows to the logarithmic phase, trypsin is used for digestion, and the cell suspension blown down by digestion is placed in a 4mL centrifuge tube. After a portion of the cell suspension and an equal volume of 0.2% trypan blue dye solution are blown and mixed, the cell count is performed to obtain the total number of cells per milliliter. After the remaining cell suspension is blown and mixed, 5000 cells per well are plated, and 100 μL of each well is added to a 96-well plate, shaken by the "cross shaking method", and then placed in a 37°C, 5% CO2 cell culture incubator for 24 hours to wait for the cells to adhere to the wall and grow out. The old culture medium is discarded with a pipette, and 100 μL of culture medium containing 0.1-1 μM lignoceric acid is added in sequence and continued to be cultured for 12 hours, and then replaced with fresh complete culture medium. Subsequently, 20 μL of MTT solution (5 mg / mL) is added and blown along the wall to each well, operated in dark, and then incubated for 3.5-4 hours. After the incubation is completed, the liquid is accurately aspirated with a spray gun, and 150 μL of DMSO solution is added to each well. The 96-well plate is placed on a low-speed shaker and shaken slowly horizontally for 10-15 minutes. After the crystals are completely dissolved and evenly mixed, the absorbance value of each well at a wavelength of 490 nm is detected using a multifunctional microplate reader. The results are as follows: Figure 1 .

[0035] according to Figure 1The results show that the concentration of lignoceric acid within 0.7 μM has no significant effect on the cell viability of DBT-1 cells.

[0036] 2. Effects of lignoceric acid on RNA virus replication under different experimental conditions

[0037] 2.1 Effects of different concentrations of pyroligneous acid on the replication of NDV-GFP and VSV viruses

[0038] After the logarithmic phase cells were digested and plated in a six-well plate, each well was added with a complete medium containing different concentrations of lignoceric acid for culture, and then NDV-GFP and VSV viruses were added for virus infection experiments, and then the virus-infected cells were collected and washed. NDV-GFP virus is a recombinant NDV virus constructed by inserting the green fluorescent protein (GFP) gene into the NDV virus genome. The cells were resuspended and centrifuged, and then washed once with phosphate buffer PBS and centrifuged. Then, the corresponding PBS was added according to the amount of cell precipitation, and 4×SDS-PAGE protein loading buffer was added according to the added PBS at a ratio of 1:3, and heated in a 100℃ metal bath for 10 minutes. After the sample was cooled, a protein immunoblotting experiment was performed. According to the steps of preparing separation gel and concentrated gel, electrophoresis, transfer, blocking, incubation with primary antibody, incubation with secondary antibody and development, the corresponding results can be obtained, and the data can be analyzed by ImageJ software.

[0039] like Figure 2 As shown, the addition of 0.1, 0.2, 0.4 and 0.5 μM lignoceric acid promoted the replication of NDV-GFP virus and VSV virus infection. Figure 1 , the effect of lignoceric acid on the proliferation ability of DBT-1 cells, and the experimental concentration of lignoceric acid added in the subsequent experiment was determined to be 0.2μM.

[0040] 2.2 Effect of adding lignoceric acid on the replication ability of NDV-GFP and VSV viruses under exogenous time gradient

[0041] When DBT-1 cells grew to the logarithmic phase, the cell suspension digested with trypsin was placed in a centrifuge tube and mixed by pipetting. 6The cells were placed in a six-well plate, shaken by the "cross shaking method", and then placed in a 37°C, 5% CO2 cell culture incubator for 24 hours. After the cell density grows to 60%, the old culture medium was discarded, and after washing with PBS, a complete culture medium containing 0.2μM lignoceric acid (DMEM culture medium containing 10% fetal bovine serum) was added in sequence. After the time gradient is continued for 6, 12, 18 or 24 hours, the complete culture medium containing lignoceric acid was discarded, and after washing with PBS, a serum-free culture medium containing NDV-GFP virus and VSV virus was added, and the culture was continued for 3 hours. After that, the culture medium was discarded, and after washing with PBS, a complete culture medium containing 2% fetal bovine serum was added to continue to culture for 16-20 hours. Subsequently, the expression of GFP and VSV-G protein levels was detected by protein immunoblotting.

[0042] like Figure 3 As shown, it is shown that the addition of lignoceric acid promotes the expression of related proteins in VSV and NDV-GFP viruses most significantly at 6h and 12h, respectively, further indicating that lignoceric acid promotes the replication of NDV-GFP virus and VSV virus infection.

[0043] 2.2 Effect of exogenous addition of lignoceric acid on the transcription levels of NDV-GFP and VSV-G

[0044] After digestion, DBT-1 cells grown to the logarithmic phase were plated in a six-well plate, and complete medium containing 0.2 μM lignoceric acid was added to each well for 12 hours of culture. NDV-GFP and VSV viruses were added for virus infection experiments. Subsequently, the virus-infected cells were collected and viral RNA was extracted (using Beijing Quanshijin TransZol Up Kit, Catalog No.: ET111-01-V2, adding solutions and performing related operations according to the instructions), reverse transcription (using Norwegian HiScript IIIRT SuperMix for qPCR (+gDNA wiper) Kit, Catalog No.: R323-01, adding solutions and performing related operations according to the instructions) and RT-qPCR experiments. The primers were diluted with ultrapure water to a final concentration of 10 μmol / L and stored at -20°C. The cDNA product and primer dilution solution were taken out, and the whole process was carried out on an ice box. Accurately pipette and prepare a mixture of 10 μL of 2xAceQUniversal SYBR qPCR Master Mix solution, 0.4 μL of upstream primer solution, 0.4 μL of downstream primer solution, 2 μL of cDNA template, and 7.2 μL of ddH20 on the eight-tube concatenation tube, and mix with a pipette to obtain a total system of 20 μL of mixed solution. Centrifuge at high speed in an instant centrifuge to ensure that there is no residue on the tube wall and avoid light. Set the parameters and program on the fluorescence quantitative PCR instrument.

[0045] The qPCR program is as follows: 1. Pre-denaturation: 95℃5min; 2. Cyclic reaction: 95℃10s, 60℃30s, 40 cycles; 3. Melting curve: 95℃15s, 60℃60s, 95℃15s; Among them, the Ct value is preferably in the range of 15-35, the baseline is usually in 3-15 cycles, 3 technical repeats are required, and the repeat difference does not exceed 0.5.

[0046] The sequences of viral protein primers are shown in Table 1.

[0047] Table 1. Primer sequences for amplifying virus-related proteins

[0048]

[0049] like Figure 4 As shown in the figure, after virus infection, exogenous addition of lignoceric acid can significantly increase the expression of NDV-GFP and VSV-G mRNA levels, which further indicates that lignoceric acid promotes the replication of NDV-GFP and VSV virus infection.

[0050] 2.4 Effects of lignoceric acid on cytokine and immune gene expression before and after RNA virus infection

[0051] Viral infection can cause innate immune response, which is specifically reflected in the activation of related signaling pathways and the production of cytokines to resist viral infection. Therefore, by detecting related cytokines and immune genes, it is shown whether the exogenous addition of lignoceric acid weakens the innate immune response, so as to increase viral amplification. Logarithmic phase DBT-1 cells were digested and plated in six-well plates, and each well was cultured with complete medium containing 0.2 μM lignoceric acid, and then NDV-GFP and VSV viruses were added for virus infection experiments. After the infection was completed, the cells were washed with PBS and viral RNA was extracted, reverse transcribed, and RT-qPCR experiments were performed (experimental steps are the same as above 2.3). The relevant primer sequences are shown in Table 2. The experimental data were statistically analyzed using GraphPad software.

[0052] Table 2. Primer sequences for cytokines and immune genes

[0053]

[0054] The results are as follows Figure 5 As shown, after NDV-GFP or VSV virus infection, exogenous addition of lignoceric acid can significantly reduce the transcription levels of intracellular RIG-1, ISG15, IFIT1, IFIT2, MX1, TNFα and IFNβ, indicating that lignoceric acid can reduce the intracellular innate immune antiviral response, thereby promoting intracellular viral infection and further improving the RNA virus replication ability.

[0055] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for efficiently amplifying RNA viruses by adding lignoceric acid, characterized in that: The method comprises the steps of exogenously adding pyroacetic acid to a culture medium of eukaryotic cells, and then infecting the eukaryotic cells with RNA viruses to promote efficient amplification of the RNA viruses.

2. The method for efficiently amplifying RNA viruses by adding lignoceric acid according to claim 1, characterized in that: The RNA virus is Newcastle disease virus NDV or vesicular stomatitis virus VSV.

3. The method for efficiently amplifying RNA viruses by adding lignoceric acid according to claim 1, characterized in that: The added concentration of the lignoceric acid is 0.1-0.5 μM.

4. The method for efficiently amplifying RNA viruses by adding lignoceric acid according to claim 1, characterized in that: The wood vinegar reduces the transcription levels of RIG-1, ISG15, IFIT1, IFIT2, MX1, TNFα and IFNβ in eukaryotic cells, thereby reducing the innate immune antiviral response in the cells, thereby promoting intracellular viral infection and improving the replication ability of RNA viruses.

5. The method for efficiently amplifying RNA viruses by adding pyroligneous acid according to claim 1, characterized in that: The time for exogenous treatment of cells with wood acetate is 6-48h.

6. A method for efficiently amplifying RNA viruses by adding pyroligneous acid according to claim 1, characterized in that: The eukaryotic cells are DBT-1 cells.

7. The method for efficiently amplifying RNA viruses by adding lignoceric acid according to claim 1, characterized in that: The virus infection time of the lignoceric acid in promoting RNA virus replication is 16-20 hours.

8. Use of the method for efficiently amplifying RNA viruses by adding lignoceric acid as claimed in any one of claims 1 to 7 in the production and development of anti-RNA virus vaccines or candidate drugs.

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