Application of 1-methylinosine in preparation of medicine for preventing and treating bombyx mori blood-type pyosis

Abstract

The invention discloses application of 1-methylinosine in preparation of a medicine for preventing and treating silkworm pyohematosis, and compared with the prior art, the application of 1-methylinosine in preparation of the medicine for preventing and treating silkworm pyohematosis has the advantages that (1) a new application form of 1-methylinosine is provided and is used for preparing the medicine for preventing and treating silkworm pyohematosis; (2) when the final use concentration of the 1-methylinosine is more than 1 mu M, the complete inhibition of the replication or proliferation of the BmNPV can be realized, and the pre-infection (prevention) and post-infection (treatment) of the BmNPV are not limited; (3) a new thought is provided for research and development of drugs for preventing and treating silkworm pyohematosis, and the application and popularization value is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a method for preventing and treating a model insect viral disease, in particular to application of 1-methylinosine in preparing a drug for preventing and treating blood-type pus disease of silkworm. Background Art

[0002] Silkworm blood pus disease is a serious infectious silkworm disease caused by Bombyx morinucleopolyhedrovirus (BmNPV), and is one of the most common diseases in silkworm breeding. Because it occurs at all stages of silkworm development and there is no obvious external infection during the infection period before the onset of the disease, it often leads to large-scale infection when it is discovered. BmNPV belongs to the family of Baculoviridae and is a type of enveloped DNA virus that specifically infects arthropods. During the virus replication process, two types of virus particles are formed, namely, occlusion derived virions (ODV) and budded virions (BV). After ODV ingested through food or body wall wounds infects the midgut epithelial columnar cells and causes primary infection, the produced BV secondarily infects other sensitive tissues and cells, causing horizontal transmission, and eventually leading to systemic infection of the host. The corpses, pus, and contaminated silkworm feces, silkworm tools, and cocoons of dead diseased silkworms, pupae, and moths also carry a large amount of viruses and are highly contagious, serving as the main source of infection for blood-type purulent disease.

[0003] At present, there is no specific drug for the treatment of blood-type pus disease in silkworms. In production, chemical disinfectants are mainly used to disinfect the entire silkworm breeding cycle to prevent the infection and spread of the virus, but these measures can only play a disinfection and prevention role. In the actual breeding process, the incubation period of blood-type pus disease in silkworms is short, the onset is fast, and the infection is difficult to detect with the naked eye. When the naked eye finds it, it is often in the late stage of the disease or the outbreak period. Once the virus infects the silkworm, the silkworm farmers are completely helpless.

[0004] In the prior art, experimental studies have shown that compounds such as β-propiolactone and nalidixic acid have a certain inhibitory effect on the incidence of BmNPV infection in silkworms, but their main role is still in prevention rather than treatment. 1-Methylinosine is an organic compound composed of purine and nucleoside, derived from inosine, and plays a similar role to nucleoside in the DNA synthesis process. Those skilled in the art often use the above-mentioned nucleoside analogs to prevent viruses from replicating in infected cells. At present, there are no patents or literature reports on the application of 1-methylinosine in the prevention and treatment of viruses and BmNPV. Summary of the invention

[0005] Technical problem to be solved: In order to overcome the deficiencies of the prior art and obtain a drug for silkworm blood-type pus disease that has both preventive and therapeutic functions, the present invention provides the use of 1-methylinosine in the preparation of a drug for preventing and treating silkworm blood-type pus disease.

[0006] Technical solution: Application of 1-methylinosine in the preparation of drugs for preventing blood-type pus disease in silkworms.

[0007] Application of 1-methylinosine in preparing medicine for treating blood-type pus disease in silkworm.

[0008] Application of 1-methylinosine in inhibiting BmNPV virus replication or proliferation in silkworm cells.

[0009] Preferably, the effective concentration of 1-methylinosine is 1-100 μM.

[0010] Preferably, the effective concentration of 1-methylinosine is 10 μM.

[0011] Preferably, the application method comprises the following steps:

[0012] S1. Dissolve 1-methylinosine in dimethyl sulfoxide (DMSO) to prepare a storage solution with a concentration of 10 mM;

[0013] S2. Add 1-methylinosine before BmNPV virus infection of silkworm cells

[0014] Inoculate Bombyx mori cells in a cell culture dish. After the cells adhere to the wall, add the storage solution in S1 to the culture dish to make the final concentration of the storage solution 1-100 μM, and continue to incubate at 27°C for 10-120 min; then infect the cells with BmNPV virus carrying the green fluorescent protein gene at MOI=10, culture at 27°C for 24 hours, add 10 μL of MTT solution to each well, continue to culture for 4 hours, add 100 μL of Formazan to each well, mix well, and culture at 27°C for 3-4 hours, measure the absorbance at 560-600 nm, and detect the effect of 1-methylinosine on cell activity;

[0015] S3. BmNPV virus infected silkworm cells and then added with 1-methylinosine

[0016] Bombyx mori cells were inoculated in a cell culture dish. After the cells adhered, they were infected with BmNPV virus carrying the green fluorescent protein gene at MOI=10. 1-Methylinosine stock solution was added 0, 12, 24, 48, 72, and 96 hours after infection to make the final concentration of the stock solution 1-100 μM. The cells were cultured at 27°C and the virus titer was determined by the endpoint dilution method using the cell culture fluid.

[0017] Preferably, before and after BmNPV virus infects silkworm cells, 1-methylinosine can significantly inhibit the transcriptional expression of vp39, lef-1 and pe38 genes.

[0018] Beneficial effects: (1) The present invention provides a new application form of 1-methylinosine for preparing drugs for the prevention and treatment of blood-type purulent disease in silkworms; (2) 1-methylinosine can completely inhibit the replication or proliferation of BmNPV when the final concentration is above 1 μM, and is not limited to before (prevention) and after (treatment) infection with BmNPV; (3) The present invention provides a new idea for the research and development of drugs for the prevention and treatment of blood-type purulent disease in silkworms, and has application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the effect of 1-methylinosine on the activity of silkworm cells;

[0020] Figure 2 It is the fluorescence image of virus-infected cells after the silkworm cells were treated with different concentrations of 1-methylinosine;

[0021] Figure 3 is a graph showing the effect of adding 1-methylinosine at different times on the replication or proliferation of BmNPV;

[0022] Figure 4 This is a graph showing the effect of different concentrations of 1-methylinosine on the BmNPV viral genome copy number;

[0023] Figure 5 This is a diagram showing the effect of 1-methylinosine on BmNPV viral gene transcription. DETAILED DESCRIPTION

[0024] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0025] The professional names and their English abbreviations involved in the embodiments of the present invention are as follows:

[0026] DMSO: dinitrosulfoxide; MTT: 3-2,5-diphenyltetrazolium bromide; Formazan: formazan; vp39: BmNPV capsid protein gene; lef-1: BmNPV late expression factor gene; pe38: BmNPV very early protein gene; ie-1: BmNPV very early expression gene.

[0027] Example 11-Effect of methylinosine on the activity of silkworm cells

[0028] This example is used to investigate the effect of 1-methylinosine on the activity of silkworm cells, and specifically comprises the following steps:

[0029] 1. Cell inoculation and preparation of 1-methylinosine storage solution

[0030] a. Inoculate approximately 10 cells in each 35 mm cell culture dish (Corning). 5 BmN cells, each cell dish contains 1 mL of TC100 insect cell culture medium with 10% FBS;

[0031] b. Culture at 27°C for 12-24 hours, and use for subsequent experiments after the cells adhere to the wall;

[0032] c. Prepare a 10 mM 1-methylinosine stock solution using DMSO.

[0033] 2. Effect of 1-methylinosine on the activity of silkworm cells

[0034] d. Add 100 μL of cells to a 96-well plate;

[0035] e. 1 μM and 10 μM 1-methylinosine were added to the experimental groups, and an equal amount of culture medium was added to the control group;

[0036] f. After 24 hours, 10 μL of MTT solution was added to each well and cultured for 4 hours;

[0037] g. Add 100 μL of Formazan solution to each well, mix well, and continue incubating in the incubator until the Formazan is completely dissolved. Incubate at 27°C for 3-4 hours.

[0038] h. Measure the absorbance at 570nm. The result is as follows Figure 1 As shown, 1-methylinosine has little effect on silkworm cells and does not affect subsequent experiments.

[0039] Example 2

[0040] This example shows the prevention and treatment of BmNPV infection carrying the egfp reporter gene at a final concentration of 1 μM 1-methylinosine:

[0041] (1) Take 10 5 Three culture dishes of cells were prepared, each containing 1 mL of 10% FBS TC100 insect cell culture medium. 0.1 μL of 10 mM 1-methylinosine stock solution and 1 μL of 10% FBS TC100 culture medium were added respectively, and cultured at 27°C for 30 min, followed by the addition of BmBac-egfp virus at an MOI of 10.

[0042] (2) After 72 hours, the three dishes were placed on a fluorescent inverted microscope to observe the expression of green fluorescent protein. Figure 2 .

[0043] (3) DNA was extracted to detect the effect of 1-methylinosine on the viral genome copy number. The results showed that when cells were treated with 1 μM 1-methylinosine, the genome copy number of BmNPV virus was reduced. Figure 4 ).

[0044] Example 3

[0045] The difference between this example and Example 2 is that the final concentration of 1-methylinosine is 10 μM. The results are as follows:

[0046] Green fluorescent protein expression is shown in Figure 2 When cells were treated with 10 μM 1-methylinosine, the fluorescence of BmNPV-infected cells was significantly reduced compared with the control, which could significantly reduce the genome copy number of BmNPV virus ( Figure 4 ).

[0047] Example 4

[0048] The difference between this example and Example 2 is that the final concentration of 1-methylinosine is 100 μM. The results are as follows:

[0049] The results showed that when cells were treated with 100 μM 1-methylinosine, the genome copy number of BmNPV virus was significantly reduced, but the copy number was higher than that at 10 μM concentration ( Figure 4 ).

[0050] Comparative Example 1

[0051] The difference between this control example and Example 2-4 is that 1-methylinosine was not added. The results are as follows: Figure 2 As shown, BmNPV-infected cells produced a lot of fluorescence.

[0052] Example 5

[0053] This example investigates the therapeutic effect of adding 1-methylinosine at different time points after BmNPV infection of cells, taking the final concentration of 1-methylinosine as 10 μM as an example:

[0054] (1) Take 10 5 Three culture dishes of cells were prepared, each containing 1 mL of TC100 insect cell culture medium containing 10% FBS. Then, BmBac-egfp virus was added at an MOI of 10; the time was set to 0 hours after infection, and 1 μL of 10 mM 1-methylinosine stock solution was added after 12, 24, 48, 72, and 96 hours, and cultured at 27°C.

[0055] (2) Take 10 μL of cell culture medium, determine the virus titer by endpoint dilution method, and draw a virus growth curve. The results show that after BmNPV completes infection of cells, adding 1-methylinosine at a final concentration of 10 μM to treat cells has a significant inhibitory effect on BmNPV, indicating that 10 μM 1-methylinosine can clear and treat BmNPV-infected cells ( Figure 3 ).

[0056] Example 6

[0057] This example investigates the effect of 1-methylinosine on gene transcription in preventing silkworm cells from being infected with BmNPV virus:

[0058] (1) Take 10 5 Three culture dishes of cells were prepared, each containing 1 mL of 10% FBS TC100 insect cell culture medium. 1 μL of 10 mM 1-methylinosine stock solution and 10 μL of 10% FBS TC100 culture medium were added, respectively, and cultured at 27°C for 30 min, followed by the addition of BmBac-egfp virus at an MOI of 10.

[0059] (2) After 48 hours, the three dishes were placed on a fluorescent inverted microscope to observe the expression of green fluorescent protein. Figure 2 Consistent.

[0060] (3) RNA was extracted and the effect of 10 μM 1-methylinosine on the transcription of genes such as vp39, lef-1, and pe38 was detected 48 hours after BmNPV infection.

[0061] The results showed that the fluorescence of BmNPV-infected cells was significantly reduced compared with the control, so 1-methylinosine had a significant inhibitory effect on BmNPV gene transcription ( Figure 5 ).

Claims

Application of 1.1-methylinosine in the preparation of drugs for preventing blood-type pus disease in silkworms. Application of 2.1-methylinosine in the preparation of drugs for treating blood-type pus disease in silkworms. Application of 3.1-methylinosine in inhibiting BmNPV virus replication or proliferation in silkworm cells.

4. The use according to any one of claims 1 to 3, characterized in that: The effective concentration of the 1-methylinosine is 1-100 μM.

5. The use according to claim 4, characterized in that: The effective concentration of 1-methylinosine is 10 μM.

6. The use according to claim 3, characterized in that: The application method comprises the following steps: S1. Dissolve 1-methylinosine in dimethyl sulfoxide (DMSO) to prepare a storage solution with a concentration of 10 mM; S2. Add 1-methylinosine before BmNPV virus infection of silkworm cells Inoculate Bombyx mori cells in a cell culture dish. After the cells adhere to the wall, add the storage solution in S1 to the culture dish to make the final concentration of the storage solution 1-100 μM, and continue to incubate at 27°C for 10-120 min; then infect the cells with BmNPV virus carrying the green fluorescent protein gene at MOI=10, culture at 27°C for 24 hours, add 10 μL of MTT solution to each well, continue to culture for 4 hours, add 100 μL of Formazan to each well, mix well, and culture at 27°C for 3-4 hours, measure the absorbance at 560-600 nm, and detect the effect of 1-methylinosine on cell activity; S3. BmNPV virus infection of silkworm cells followed by addition of 1-methylinosine Bombyx mori cells were inoculated in a cell culture dish. After the cells adhered, they were infected with BmNPV virus carrying the green fluorescent protein gene at MOI=10. 1-Methylinosine stock solution was added 0, 12, 24, 48, 72, and 96 hours after infection to make the final concentration of the stock solution 1-100 μM. The cells were cultured at 27°C and the virus titer was determined by the endpoint dilution method using the cell culture fluid.

7. The use according to claim 6, characterized in that: Before and after BmNPV virus infected silkworm cells, 1-methylinosine could significantly inhibit the transcriptional expression of vp39, lef-1 and pe38 genes.

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