Application of endoplasmic reticulum protein antioxidant in prevention and treatment of bombyx mori nuclear polyhedrosis virus

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to application of an endoplasmic reticulum protein antioxidant (reducing agent) in prevention and treatment of bombyx mori nuclear polyhedrosis virus. A screening test finds that the related endoplasmic reticulum protein antioxidants beta-mercaptoethanol (BME), dithiothreitol (DTT) and / or tris (2-carboxyethyl) phosphine (TCEP) have the effect of inhibiting the bombyx mori nuclear polyhedrosis virus (BmNPV), and the antioxidants interfere the replication and proliferation of the virus by destroying disulfide bonds in key proteins of the BmNPV, so that the bombyx mori nuclear polyhedrosis virus can be inhibited. The stability and activity of virions are directly influenced, so that the infection of the viruses on the silkworms is inhibited, a brand new thought is provided for the development of antiviral drugs of the silkworms, and the application value is good.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and in particular, the invention relates to application of endoplasmic reticulum protein antioxidant in preventing and controlling silkworm nuclear polyhedrosis virus. Background Art

[0002] Bombyx mori is an insect belonging to the genus Bombyx mori of the family Bombycidae, also known as mulberry silkworm. Bombyx mori is a holometabolous insect, and its development process is divided into four stages: egg, larva, pupa and adult. Silkworm breeding occupies an important position in the development of my country's agricultural economy and silk industry. Bombyx mori nuclear polyhedrosis virus (BmNPV) is the most harmful of the three major viral diseases in the silkworm industry. It has outbreaks in silkworm-raising countries around the world. It is highly contagious and difficult to control, often causing huge economic losses. The primary infection of NPV occurs in the midgut epithelial tissue, and the primary target cells include columnar cells and regenerative cells. When NPV enters the cell, a series of processes such as virus reproduction, packaging, and cell death occur, and the virus uses the host cell to reproduce itself. Bombyx mori has an irreplaceable position in the silk industry, but the high frequency of disease caused by BmNPV is still a bottleneck problem restricting the development of the silkworm industry. Existing prevention and control measures, such as chemical agents and traditional disease management methods, have problems such as limited effects and environmental pollution. At present, there is no specific drug reported for Bombyx mori nuclear polyhedrosis virus (BmNPV) infection. Therefore, there is an urgent need for a method that can quickly and efficiently inhibit BmNPV infection to lay a scientific foundation for studying the mechanism of silkworm resistance to BmNPV and virus prevention and control in sericulture production.

[0003] Studies have shown that viral infection can trigger the rapid synthesis of a large number of viral proteins in host cells, leading to the accumulation of misfolded proteins, further inducing endoplasmic reticulum stress and cellular oxidative damage. Antioxidation is one of the means of preventing and controlling viral infection. However, the relationship between BmNPV virus infection prevention and control and antioxidant therapy in silkworms needs further study. Summary of the invention

[0004] In order to further improve the control effect of Bombyx mori nuclear polyhedrosis virus (BmNPV) and provide ideas for the control of the virus, the present invention has been found through long-term research that antioxidants (reducing agents) targeting endoplasmic reticulum proteins can exert a good antiviral effect in silkworms, thereby completing the present invention.

[0005] In one aspect of the present invention, the present invention discloses the use of an endoplasmic reticulum protein antioxidant (reducing agent) in the preparation of a preparation for preventing and treating Bombyx mori nuclear polyhedrosis virus (BmNPV).

[0006] In one embodiment, the antioxidant (reducing agent) is β-mercaptoethanol (BME), dithiothreitol (DTT) and / or tris(2-carboxyethyl)phosphine (TCEP); more preferably, the antioxidant (reducing agent) is β-mercaptoethanol (BME).

[0007] In one aspect of the present invention, the present invention discloses a preparation for controlling Bombyx mori nuclear polyhedrosis virus (BmNPV), wherein the preparation contains β-mercaptoethanol (BME), dithiothreitol (DTT) and / or tris(2-carboxyethyl)phosphine (TCEP).

[0008] In one embodiment, the preparation is a powder, a granular preparation, or a liquid preparation. Preferably, the preparation is a liquid preparation.

[0009] In one embodiment, the preparation is applied to silkworms by feeding to achieve the effect of preventing and treating Bombyx mori nuclear polyhedrosis virus (BmNPV) infection. Preferably, the preparation is a liquid preparation, and the preparation is sprayed on mulberry leaves, the food of silkworms, for feeding.

[0010] In one embodiment, the preparation is used at a concentration of 2 mM for silkworm bodies and 2 mM for cells.

[0011] In one embodiment, the preparation is administered before the silkworm is infected with BmNPV, or within 12 hours after being infected with BmNPV.

[0012] In one embodiment, the formulation includes a pharmaceutically acceptable excipient that can enhance the stability and palatability of the drug.

[0013] A method of the present invention discloses a method for preventing and controlling Bombyx mori nuclear polyhedrosis virus (BmNPV), wherein an endoplasmic reticulum protein antioxidant (reducing agent) is applied to the silkworm; preferably, the antioxidant is β-mercaptoethanol (BME), dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), glutathione (GSH), N-acetylcysteine ​​(NAC) and sodium sulfide (Na2S); more preferably, the antioxidant (reducing agent) is β-mercaptoethanol (BME), dithiothreitol (DTT) and / or tris(2-carboxyethyl)phosphine (TCEP). Beneficial Effects

[0014] (1) Through research and testing, the present invention found that applying endoplasmic reticulum protein antioxidants (reducing agents) to silkworms can disrupt the disulfide bonds in the key proteins of BmNPV, interfere with the replication and proliferation of the virus, and directly affect the stability and activity of the virus particles, thereby inhibiting the virus from infecting the silkworm. This provides a new idea for the development of antiviral drugs for silkworms and has good application value.

[0015] (2) Reduce the burden of viral infection on host cells and improve the health and productivity of silkworms. The present invention shows that the application of BME not only effectively inhibits the proliferation of BmNPV, but also protects the functional stability of host cells by indirectly reducing the cell stress response caused by viral infection. Further studies have found that BME helps maintain protein homeostasis in silkworm cells and optimizes cell metabolism while reducing viral proteins, which is beneficial to the healthy growth of silkworms and the improvement of production performance.

[0016] (3) Excellent antiviral effect and application prospects, helping silkworm technology innovation. Compared with traditional antiviral methods, the present invention uses redox reagents as the core antiviral strategy, which is precise and efficient. By controlling the dosage and application method (such as feeding or mulberry leaf spraying), BME can play a significant inhibitory role in the early stage of viral infection and early intervention. Combined with acceptable pharmaceutical excipients, the stability and palatability of the drug are further enhanced, providing technical support for the green and sustainable development of the silkworm industry, showing extremely high development and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 , are the fluorescence intensities in silkworm BmN cells infected with BmNPV-EGFP at different time periods after treatment with six redox drugs, among which A is BME, B is TCEP, C is DTT, D is GSH, E is NAC, and F is Na2S.

[0018] Figure 2 , is a flow cytometry quantitative analysis of the viral inhibition efficiency of BmNPV-infected cells after treatment with BME, DTT, and TCEP.

[0019] Figure 3 , combined with TUNEL staining and MTT to detect the effect of BME drug treatment on BmNPV-infected cells, among which, Figure 3 In A, green fluorescence represents the presence of apoptotic DNA fragments in cells, and blue represents DAPI-stained nucleic acids. The MTT method (B) was used to evaluate the survival of cells at different time points (24 h, 48 h, and 72 h). 490 Values ​​indicate cell viability.

[0020] Figure 4 , to detect the virus that proliferates BmNPV after BME drug treatment GP64 Gene expression analysis.

[0021] Figure 5 , to detect the virus that proliferates BmNPV after BME drug treatment GP64 Effect of gene gDNA copy number.

[0022] Figure 6 2. The effect of BME drug treatment on the proliferation of BmNPV in cells was detected by detecting the viral GP64 protein. The brightness of the target protein GP64 band after BME drug treatment was detected compared with the blank control and virus infection control.

[0023] Figure 7 , to detect the effect of BME treatment on the proliferation of BmNPV in silkworms GP64 Gene expression. In the figure, A is the number of viral gDNA copies in the midgut after adding the drug, B is the number of viral gDNA copies in the hemolymph, C is the number of viral gDNA copies in the fat body, D is the expression of the viral GP64 gene in the midgut after adding the drug, E is the expression of the viral GP64 gene in the hemolymph, and F is a comparison of the expression of the viral GP64 gene in the fat body. DETAILED DESCRIPTION

[0024] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially. Example 1: Screening of antioxidant agents (reducing agents)

[0025] One day in advance, Bombyx mori BmN cells were plated in 12-well plates, and the cell density reached 80%-90% the next day. Six different reducing agents, β-mercaptoethanol (BME), dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), glutathione (GSH), N-acetylcysteine ​​(NAC) and sodium sulfide (Na2S) were added to the cell culture plate at equal concentrations (1mM, 2mM, 4mM). After 12 hours of drug addition, BmNPV virus was added, and fluorescence was taken at different time points (24h, 48h, 72h) to observe the fluorescence signal strength of EGFP-BmNPV in the cells after drug addition. See Figure 1, fluorescence microscopy showed that compared with the control group, after the BmNPV virus-infected cells were treated with the same concentration of BME, TCEP, and DTT for 24h, 48h, and 72h, the fluorescence intensity of the BmNPV virus with the GFP tag weakened, indicating that BME, TCEP, and DTT had a good inhibitory effect on the proliferation of the virus. Similarly, compared with the control group, the fluorescence intensity of the virus did not change significantly after the treatment with GSH, NAC, and Na2S. Further tests were performed with the subsequent treatment with BME, TCEP, and DTT. Example 2: Quantitative analysis of the virus inhibition efficiency of BmNPV infected cells after treatment with BME, DTT, and TCEP by flow cytometry

[0026] Using flow cytometry with DMSO as the control, the three reagents BME, DTT and TCEP were detected at a concentration of 2mM to treat cells infected with BmNPV-EGFP, and quantitative analysis was performed to evaluate the viral inhibitory effect of the relevant drugs 24 hours after addition.

[0027] See also Figure 2 Compared with the control group, the proportion of apoptotic cells in the BME-treated group was significantly lower than that in the DTT and TCEP-treated groups, indicating that BME has a strong effect in inhibiting viral infection and cell apoptosis caused by it. The cell apoptosis rate in the BME group remained at a low level, indicating that it can effectively inhibit the proliferation of BmNPV and reduce cell damage caused by viral infection.

[0028] In contrast, the proportion of apoptotic cells in the DTT and TCEP treatment groups was higher, especially in the TCEP group, where the number of apoptotic cells did not decrease significantly, indicating that its inhibitory effect on the virus was weak. The DTT treatment group also failed to significantly reduce the proportion of apoptotic cells after 24 hours, which may be due to the limitations of its antiviral effect. In view of this, BME showed a significant antiviral effect in this experiment and was able to effectively inhibit cell apoptosis caused by BmNPV infection, while the effects of DTT and TCEP were relatively weak. Example 3 Effects of BME drug treatment on BmNPV-infected cells

[0029] TUNEL staining was used to detect cell apoptosis after BME treatment, where green fluorescence represents the presence of apoptotic DNA fragments in cells and blue represents DAPI-stained nucleic acids.

[0030] See also Figure 3, TUNEL staining results showed: In the Control (BmNPV-) control group not infected with BmNPV, no green fluorescence was observed by TUNEL staining, indicating that no obvious apoptosis occurred in the cells. In the Control (BmNPV+) group, i.e. the BmNPV-infected group (untreated BME), green fluorescence was clearly visible, indicating that BmNPV infection caused cell apoptosis. In the BME-treated infected group (BME (BmNPV+)), green fluorescence was significantly reduced, and the number of apoptotic cells was significantly decreased compared with the untreated group. This shows that BME can alleviate cell apoptosis caused by BmNPV infection.

[0031] The MTT method was further used to evaluate the survival of cells at different time points (24 h, 48 h, and 72 h), and the cell activity was expressed by the OD490 value. The MTT test results showed that at 24 hours after infection (24 hpi), there was no significant difference in the OD490 values ​​between the BME-treated group and the untreated group, indicating that BME had little effect on cell activity in the early stage of viral infection. At 48 hours (48h.pi) and 72 hours (72 hpi) of infection, the OD490 value of the BME-treated group was significantly higher than that of the untreated group (Control, BmNPV+). This shows that BME treatment can effectively increase the activity of cells in the late stage of BmNPV infection and reduce the damage of the virus to cells.

[0032] In summary, TUNEL staining results showed that BME treatment could significantly inhibit cell apoptosis caused by BmNPV infection; at the same time, MTT assay showed that BME could significantly increase the survival rate of BmNPV-infected cells. These results indicate that BME has the effect of anti-viral proliferation and protecting cell activity. Example 4: Effect of BME drug treatment on BmNPV proliferation

[0033] Well-growing BmN cells were selected and infected with BmNPV for treatment. They were divided into a control group (DMSO) and an experimental group (BME) and treated with different concentration gradients of 1, 2, and 4 mM for 24 h, 48 h, and 72 h. GP64 mRNA, reverse transcribe to synthesize cDNA and use RT-qPCR to detect viral gene expression. Figure 4 The results showed that compared with the control group, BME can effectively downregulate viral gene expression and the inhibitory effect will become more obvious as time goes by. At the same time, there is no significant difference in the effect of BME concentration of 2mM and 4mM on viral replication. Therefore, it can be judged that in in vitro experiments, BME concentration of 2mM has the best inhibitory effect on Bombyx mori BmNPV virus.

[0034] Furthermore, the changes in the gDNA copy number of the GP64 gene of BmNPV proliferation after 24h, 48h, and 72h of treatment with 2mM BME were detected. Using an equal volume of culture medium as a control, the virus particles BV in the supernatant separated by centrifugation were detected by collecting cells. GP64 For gene copy number, see Figure 5 Based on the exported RT-qPCR data, the results were analyzed by EXCEl worksheet calculation and GraphPad Prism 9.3.1 drawing. The results showed that compared with the Control, BME effectively inhibited the viral gDNA copy as the time gradient changed, further indicating that the reducing agent BME has a good effect in inhibiting the replication of the silkworm BmNPV virus. Example 5 Effect of BME Drug Treatment on Detection of Viral GP64 Protein in Virus-Infected Cells

[0035] The expression level of viral GP64 protein was detected by Western blot to evaluate the effect of BME drug treatment on viral GP64 protein in BmN cells infected with BmNPV. GP64 is the main envelope protein of BmNPV, and its expression level can reflect the replication and proliferation of the virus in host cells. β-Tubulin was used as an internal reference to verify the consistency of the loading amount of each sample. Figure 6 , the intensity of β-Tubulin bands in each experimental group was basically the same, indicating that the loading amount of the sample was uniform and the experimental results were reliable. In the control group (Control +), the expression level of GP64 protein was significantly higher in the infection group without BME treatment (+ BmNPV, - BME), indicating that BmNPV has active replication and proliferation in cells. In the BME treatment group (Control +, + BME), the expression level of GP64 protein was significantly reduced in the infection group treated with BME (+ BmNPV, + BME), and the expression intensity was significantly weakened compared with the untreated group, indicating that BME treatment can effectively inhibit the proliferation of BmNPV. In the non-infected group (Control -), the expression of GP64 protein was not detected in the group not infected with BmNPV (- BmNPV, ± BME), further proving that GP64 protein is specifically associated with viral replication. Western Blot results showed that BME treatment can significantly reduce the expression level of GP64 protein in BmNPV-infected cells, indicating that BME has a strong inhibitory effect on the replication and proliferation of BmNPV. Example 6 Detection of viral GP64 gene in silkworms infected with BmNPV by BME treatment

[0036] The effect of adding 1mM and 2mM BME on BmNPV virus replication was detected in 5th instar silkworms with good growth. The gDNA copy number of BmNPV GP64 in the midgut, fat body and hemolymph of silkworms was detected by qPCR using gDNA template, see Figure 7 , and found that the copy number after knockdown was significantly lower than that of the control group, indicating that BME treatment inhibited the replication of the virus. Subsequently, the gene expression of viral GP64 in the midgut, fat body and hemolymph of the p50 strain of silkworms infected with BmNPV was detected. Compared with the control group, the mRNA expression of the experimental group was significantly downregulated, and the treatment effect of BME 2mM was better, which further verified the inhibitory effect of BME on viral replication.

[0037] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of endoplasmic reticulum protein antioxidant (reducing agent) in the preparation of preparations for the prevention and treatment of Bombyx mori nuclear polyhedrosis virus (BmNPV).

2. The use according to claim 1, characterized in that: The antioxidant (reducing agent) is β-mercaptoethanol (BME), dithiothreitol (DTT) and / or tris(2-carboxyethyl)phosphine (TCEP); more preferably, the antioxidant (reducing agent) is β-mercaptoethanol (BME).

3. The use according to claim 1, characterized in that: The preparation is a powder, a granular preparation or a liquid preparation. Preferably, the preparation is a liquid preparation.

4. A preparation for preventing and treating Bombyx mori nuclear polyhedrosis virus (BmNPV), characterized in that: The formulation contains β-mercaptoethanol (BME), dithiothreitol (DTT) and / or tris(2-carboxyethyl)phosphine (TCEP).

5. The preparation according to claim 4, characterized in that The preparation is a powder, a granular preparation or a liquid preparation. Preferably, the preparation is a liquid preparation.

6. The preparation according to claim 4, characterized in that The preparation is applied to silkworms by feeding to achieve the effect of preventing and treating Bombyx mori nuclear polyhedrosis virus (BmNPV) infection; preferably, the preparation is a liquid preparation, and the preparation is sprayed on mulberry leaves, the food of the silkworms, for feeding.

7. The preparation according to claim 4, characterized in that The preparation is used at a concentration of 2 mM for silkworm bodies and 2 mM for cells.

8. The preparation according to claim 4, characterized in that The preparation is administered before the silkworm is infected with BmNPV, or within 12 hours after being infected with BmNPV.

9. The preparation according to claim 4, characterized in that The preparation also includes pharmaceutically acceptable excipients.

10. A method for preventing and controlling Bombyx mori nuclear polyhedrosis virus (BmNPV), characterized in that: Applying an endoplasmic reticulum protein antioxidant (reducing agent) to silkworms; preferably, the antioxidant is β-mercaptoethanol (BME), dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), glutathione (GSH), N-acetylcysteine ​​(NAC) and sodium sulfide (Na2S); more preferably, the antioxidant (reducing agent) is β-mercaptoethanol (BME), dithiothreitol (DTT) and / or tris(2-carboxyethyl)phosphine (TCEP).

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