Bombyx mori nuclear polyhedrosis virus resisting medicine and application thereof

Abstract

The invention discloses a bombyx mori nuclear polyhedrosis virus resisting medicine and application thereof. Experimental results show that artesunate and dihydroartemisinin have the effect of resisting the bombyx mori nuclear polyhedrosis virus, can limit proliferation of viruses in cells by inhibiting cell autophagy induced by the bombyx mori nuclear polyhedrosis virus, can be used as a novel medicine for resisting the bombyx mori nuclear polyhedrosis virus of bombyx mori, and can be used for preparing a novel medicine for resisting the bombyx mori nuclear polyhedrosis virus of bombyx mori. The medicine is provided for preventing and treating the silkworm pyohematosis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a drug for resisting Bombyx mori nuclear polyhedrosis virus and application thereof. Background Art

[0002] Bombyx mori NPV (BmNPV) is the pathogen of blood-type purulent disease of silkworm, which has the characteristics of rapid onset and difficulty in control, and there is still no effective prevention and treatment drug so far. Therefore, it is urgent to find new and effective anti-BmNPV drugs. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an anti-Bombyx mori nuclear polyhedrosis virus drug and its application.

[0004] The first object of the present invention is to provide the use of artesunate in preparing a drug for resisting blood-type pus disease of silkworm.

[0005] Preferably, the drug against Bombyx mori blood-type purulent disease is a drug against Bombyx mori nuclear polyhedrosis virus.

[0006] Preferably, the anti-Bombyx mori nuclear polyhedrosis virus drug inhibits the proliferation of Bombyx mori nuclear polyhedrosis virus by inhibiting cellular autophagy.

[0007] The second object of the present invention is to provide the use of dihydroartemisinin in the preparation of drugs against blood-type purulent disease of silkworms.

[0008] Preferably, the drug against Bombyx mori blood-type purulent disease is a drug against Bombyx mori nuclear polyhedrosis virus.

[0009] Preferably, the anti-Bombyx mori nuclear polyhedrosis virus drug inhibits the proliferation of Bombyx mori nuclear polyhedrosis virus by inhibiting cellular autophagy.

[0010] The present invention has the following beneficial effects:

[0011] The experimental results of the present invention show that artesunate and dihydroartemisinin can inhibit the proliferation of Bombyx mori nuclear polyhedrosis virus, and artesunate and dihydroartemisinin inhibit the autophagy caused by Bombyx mori nuclear polyhedrosis virus infection of silkworm ovarian cells. Artesunate and dihydroartemisinin have the effect of resisting Bombyx mori nuclear polyhedrosis virus, and can limit the proliferation of the virus in cells by inhibiting the cell autophagy induced by Bombyx mori nuclear polyhedrosis virus. They can be used as a new drug for silkworms to resist Bombyx mori nuclear polyhedrosis virus, and provide drugs for preventing and treating blood-type purulent disease of silkworms. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1CCK8 was used to detect the effects of different concentrations of drugs on the growth of BmN cells; Note: The data in the figure are mean ± SD, n = 4; *p < 0.05, the difference is significant; **p < 0.01, ***p < 0.005, the difference is extremely significant.

[0013] Figure 2 The figure shows the effects of different drugs on the proliferation of EGFP-BmNPV in BmN cells.

[0014] Figure 3 The inhibitory effect of drug 1 on autophagy in BmN cells was detected by WB.

[0015] Figure 4 is the effect of drug 1 treatment on BmNPV proliferation in BmN cells; Note: a: The effect of 10 μmol / L drug 1 treatment on BmNPV proliferation was observed under a fluorescence microscope; b: Western Blot detection of the effect of drug 1 on cell autophagy and viral EGFP-BmNPV proliferation; c: Virus titer at different time points after drug 1 treatment; d: Grayscale scanning data analysis of the Western Blot detection results corresponding to b.

[0016] Figure 5 The effect of drug 3 on BmNPV proliferation in BmN cells; Note: a: The effect of 10 μmol / L drug 3 on BmNPV proliferation was observed under a fluorescence microscope; b: Western Blot detection of the effect of drug 3 on cell autophagy and viral EGFP-BmNPV proliferation; c: Grayscale scanning data analysis of the Western Blot detection results corresponding to b. DETAILED DESCRIPTION

[0017] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0018] In the following examples, the experimental drugs are drug 1 (artesunate), drug 2 (artemisinin), drug 3 (dihydroartemisinin), and drug 4 (artemether). The results show that drug 1 and drug 3 can inhibit the autophagy of BmN cells and inhibit the proliferation of BmNPV, while drug 2 and drug 4 have no significant inhibitory effect on BmNPV. The above results show that: for silkworm BmN cells, drugs 1 and 3 both have anti-BmNPV effects, providing a theoretical basis for the subsequent screening of new anti-BmNPV drugs.

[0019] Example 1

[0020] 1. Experimental Methods

[0021] 1. CCK8 method to test the toxicity of drugs to BmN cells (silkworm ovary cells)

[0022] Cell Counting Kit-8 (CCK-8) was used to detect the toxicity of drugs to BmN cells. The specific steps are as follows:

[0023] (1) 100 μL (about 2000) BmN cells were added to a 96-well cell culture plate, and 3 μL of drugs of different concentrations were added. Eight wells were treated with each drug, and a DMSO control was set up. The plates were cultured continuously at 28°C for 48 h.

[0024] (2) Add 10 μL of CCK-8 solution to each well in a light-proof environment.

[0025] (3) Incubate at 28°C for 3.5 h.

[0026] (4) Measure absorbance at a wavelength of 450 nm.

[0027] 2. Preparation of EGFP-BmNPV recombinant virus

[0028] The Bac-to-Bac system was used to construct a BmNPV recombinant virus with green fluorescence. The recombinant transfer vector pFastBacHTb-EGFP was transformed into Escherichia coli DH10Bac competent cells containing the BmNPV genome, and the recombinant bacmid Bacmid-EGFP was obtained through resistance screening and PCR identification. When the BmN cell culture grew to 40% to 50% of the six-well plate, the recombinant bacmid Bacmid-EGFP was transfected into the BmN cells, and the supernatant collected 72 hours after transfection was the EGFP-BmNPV recombinant virus.

[0029] 3. Proliferation Detection of Recombinant Virus EGFP-BmNPV

[0030] (1) Fluorescence microscopy: The recombinant virus EGFP-BmNPV carries the fluorescent reporter gene EGFP. Green fluorescence is observed under a fluorescence microscope, and the density of green fluorescence displayed can reflect the proliferation of the virus.

[0031] (2) Western blot method: The Western blot method was used to detect viral proliferation using EGFP monoclonal antibody.

[0032] (3) Virus titer determination: The virus titer was determined by the TCID50 method. The specific operation method is as follows: When the BmN cells were cultured in a 96-well plate to a growth density of about 80%, the culture medium was discarded and 10-fold serial dilutions (10 -1 ~10 -12) of the recombinant virus EGFP-BmNPV dilution, 100 μL each, each dilution set 8 replicates. After culturing at 28 ° C for 5 days, use an inverted fluorescence microscope to observe, record the number of wells with and without virus in the cell culture wells infected with each dilution, and calculate TCID50 at a dilution of about 50% infection "critical value". Karber method to calculate virus titer (TCID50): lgTCID50 = Ld (s-0.5), where L = the logarithm of the highest dilution, d = the difference between the logarithms of the dilutions, and s = the sum of the positive well ratios.

[0033] 4. Detection of Cellular Autophagy Levels

[0034] Western Blot was used to detect the content of autophagy marker ATG8-PE, and the ATG8-PE / ATG8 ratio was used to measure the level of autophagy in cells.

[0035] 2. Experimental Results

[0036] 1. Drug toxicity to BmN cells

[0037] In order to detect whether the drug is toxic to BmN cells, the CCK8 kit was used to analyze the effect of the drug on cell proliferation. The experimental results showed that drug 1 had no significant effect on cell proliferation at a concentration of 10 μmol / L, inhibited cell proliferation at a concentration of 20 μmol / L, and showed obvious toxicity; drug 2 had no significant effect on cell proliferation at a concentration of 50 μmol / L, and showed obvious cytotoxicity at a concentration of 100 μmol / L; drug 3 had no significant effect on cell proliferation at a concentration of 30 μmol / L, and showed cytotoxicity at a concentration of 50 μmol / L; drug 4 had no cytotoxicity at a concentration of 10 μmol / L, and showed cytotoxicity at a concentration of 20 μmol / L ( Figure 1 ).

[0038] 2. Inhibitory effect of drugs on the proliferation of EGFP-BmNPV

[0039] The effects of the four drugs on the proliferation of BmNPV were tested at a concentration of 10 μmol / L. Figure 2 As shown. After 48 hours of drug treatment, we found that drug 1 and drug 3 had a significant inhibitory effect on BmNPV proliferation, while drug 2 and drug 4 had no significant inhibitory effect on BmNPV proliferation. We selected drug 1 and drug 3 to further explore their inhibitory effects on BmN cells.

[0040] 3. Drug 1 and Drug 3 inhibit autophagy induced by BmNPV infection in BmN cells

[0041] Drug 1 and drug 3 have an inhibitory effect on the proliferation of BmNPV. We further verified whether drug 1 and drug 3 affect the autophagy level of host cells. The PE-modified band of ATG8 was detected by Western blot. The results were ( Figure 3 ) found that: a weaker BmATG8-PE band was detected in normally cultured BmN cells, indicating that a low level of autophagy occurred in normally cultured BmN cells; after treatment with 5, 10, 20, and 50 μmol / L drug 1 for 24 hours, the BmATG8-PE band weakened with the increase in drug concentration, indicating that drug 1 inhibited autophagy in BmN cells, and the inhibition of autophagy increased with the increase in concentration within the range of 5-50 μmol / L.

[0042] When EGFP-BmNPV-infected BmN cells were treated with 10 μmol / L of drug 1, and simultaneously infected with virus and treated with drug, the proliferation of virus was observed by fluorescence microscopy at 24h, 36h and 48h, and it was found that drug 1 could significantly inhibit the proliferation of virus ( Figure 4 a); Western blot results showed that after drug 1 treatment, the PE modification of BmATG8 protein decreased, indicating that drug 1 inhibited the autophagy level of BmNPV-infected cells ( Figure 4 b, d); TCID at different time points 50 The virus titer TCID was shown after 48 h of drug 1 treatment. 50 Significantly lower than DMSO control ( Figure 4 c) in.

[0043] When EGFP-BmNPV-infected BmN cells were treated with 10 μmol / L of drug 3, and simultaneously infected with virus and treated with drug, the proliferation of virus was observed by fluorescence microscopy at 24h, 36h and 48h, and it was found that drug 3 could significantly inhibit the proliferation of virus ( Figure 5 a); Western blot results showed that drug 3 inhibited BmNPV infection-induced cellular autophagy ( Figure 5 b, c) in the above.

[0044] The above experimental results show that drugs 1 and 3 have antiviral effects on silkworm BmNPV and can be used as new drugs for silkworms to resist BmNPV.

Claims

1. The application of artesunate in the preparation of drugs against blood-type purulence of silkworms.

2. The use according to claim 1, characterized in that: The anti-silkworm blood-type pus disease drug is an anti-silkworm nuclear polyhedrosis virus drug.

3. The use according to claim 2, characterized in that: The anti-Bombyx mori nuclear polyhedrosis virus drug inhibits the proliferation of Bombyx mori nuclear polyhedrosis virus by inhibiting cell autophagy.

4. Application of dihydroartemisinin in the preparation of drugs against silkworm blood-type purulence.

5. The use according to claim 4, characterized in that: The anti-silkworm blood-type pus disease drug is an anti-silkworm nuclear polyhedrosis virus drug.

6. The use according to claim 5, characterized in that: The anti-Bombyx mori nuclear polyhedrosis virus drug inhibits the proliferation of Bombyx mori nuclear polyhedrosis virus by inhibiting cell autophagy.

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