Application of tunicamycin 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 tunicamycin to prevention and treatment of bombyx mori nuclear polyhedrosis viruses. Bioinformatics analysis, cell transfection gene overexpression, gene knockout, apoptosis detection and other test methods find that bombyx mori GPT (BmGPT) plays an important role in BmNPV proliferation, at least part of BmGPT knock-down inhibits BmNPV proliferation by enhancing BmN cell apoptosis, the GPT inhibitor chicamycin can inhibit BmNPV proliferation in bombyx mori cells, and the BmNPV proliferation inhibitor can inhibit BmNPV proliferation in bombyx mori cells. And a new means is provided for prevention and treatment of the BmNPV of the silkworms.

Description

Technical Field

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

[0002] Mulberry and silkworm (Bombyx mori) are economically important animals that have long been widely used in global agriculture. During silkworm rearing, many types of pathogens infect silkworms; therefore, silkworms are a good model for studying the molecular mechanisms of host-pathogen interactions. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the most intensively studied baculoviruses, which has a large and supercoiled double-stranded DNA genome packaged in baculovirus particles and poses a major threat to the silkworm industry. Silkworm diseases caused by BmNPV infection cause a large loss of 20-50% in silkworm cocoon production in the silkworm industry every year. BmNPV produces budding virus (BV) and occluded derivative virus (ODV) during its life cycle. BV is required for intercellular infection of infected larvae. ODV is packaged to form occluded bodies (OBs) and released into the environment to infect other individual larvae. Although some virus-resistant silkworm varieties have been developed in recent years and some antiviral drugs have been discovered, the control of BmNPV in silkworms remains a huge challenge.

[0003] N-acetylglucosamine-1-phosphotransferase (GPT), also known as UDP-N-acetylglucosamine, polyterpene phosphate N-acetylglucosamine-1-P transferase (GPT), is an endoplasmic reticulum-resident transmembrane protein that catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine in the cytoplasm to polyterpene phosphate on the cytoplasmic side of the endoplasmic reticulum membrane. This is one of the initial steps in the polyterpene-linked oligosaccharide biosynthesis and protein N-glycosylation pathways. GPT regulates a variety of biological processes, such as endoplasmic reticulum protein folding, secretory protein stability, receptor binding, viral budding, and viral infectivity. The activity of GPT has been verified in eukaryotic cells, and the antibacterial agent tunicamycin has been verified to be able to inhibit the activity of GPT. However, it is still unclear whether the silkworm GPT is involved in regulating the proliferation of BmNPV. Whether tunicamycin can serve as an inhibitor of silkworm BmNPV needs further exploration. Summary of the invention

[0004] Based on the general demand for preventing and controlling Bombyx mori nuclear polyhedrosis virus and reducing the huge impact of the virus on the silkworm industry, the present invention has found through long-term and in-depth research that GPT in silkworms is also involved in the infection process of BmNPV and tunicamycin can play a role in inhibiting infection, thus completing the present invention.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides the use of tunicamycin in preventing and controlling Bombyx mori nuclear polyhedrosis virus.

[0007] In a second aspect of the present invention, the present invention provides the use of tunicamycin in the preparation of a preparation for preventing and treating Bombyx mori nuclear polyhedrosis virus.

[0008] In a third aspect of the present invention, a method for preparing a preparation for controlling Bombyx mori nuclear polyhedrosis virus is provided, wherein the preparation comprises tunicamycin.

[0009] In one embodiment, the reagent further contains a solvent required to dissolve tunicamycin, and preferably the solvent includes one or more of glycerol, propylene glycol, aldehyde alcohol, and DMSO.

[0010] In one embodiment, the tunicamycin content of the formulation is 1-10%.

[0011] In one embodiment, the preparation is administered to silkworms by oral feeding, and the administration concentration is 0.01%; for example, the preparation solution is sprayed on the mulberry leaves of silkworms and then fed to the silkworms to achieve the purpose of prevention and control.

[0012] In one embodiment, the formulation is in the form of an emulsion.

[0013] The present invention identified a GPT homolog (BmGPT) in the silkworm genome. Spatiotemporal expression profiles showed that BmGPT was highly expressed in most organs of silkworms. BmGPT regulates a variety of cellular processes, such as cell activity, proliferation and cell death. In addition, overexpression of BmGPT increased the proliferation of BmNPV in silkworm BmN cells, while inhibition of BmGPT by siRNA or chemical inhibitors inhibited the proliferation of BmNPV, both in vitro and in vivo. These results indicate that BmGPT promotes the proliferation of BmNPV by regulating cell proliferation and apoptosis, while tunicamycin, an inhibitor of GPT, can inhibit the infection of BmNPV.

[0014] Beneficial Effects

[0015] Through analysis and testing, the present invention finds that Bombyx mori GPT (BmGPT) plays an important role in the proliferation of BmNPV, and knocking down BmGPT inhibits the proliferation of BmNPV at least in part by enhancing the apoptosis of BmN cells. The use of tunicamycin, an inhibitor of GPT, can inhibit the proliferation of BmNPV in silkworm cells, providing a new approach for the prevention and treatment of BmNPV in silkworm. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 , the induced expression pattern of BmGPT gene in time and space.

[0017] Figure 2, BmGPT gene regulates the activity, proliferation and apoptosis of BmN cells.

[0018] Figure 3 , overexpression of BmGPT promoted the proliferation of BmNPV in BmN cells.

[0019] Figure 4 , BmGPT knockdown inhibited the proliferation of BmNPV in BmN cells.

[0020] Figure 5 , BmGPT knockdown promoted the apoptosis of BmN cells.

[0021] Figure 6 , apoptosis are involved in the regulation of BmNPV proliferation by BmGPT.

[0022] Figure 7 Knocking down the BmGPT gene in silkworms reduced the amplification of BmNPV.

[0023] Figure 8 , tunicamycin can reduce the BmNPV gDNA copy number and viral G64 protein level.

[0024] Fig. 9 Specific inhibition of BmGPT in silkworms can reduce the proliferation of BmNPV. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in the embodiments and test examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] Embodiment 1:

[0027] 1. Test Method

[0028] 1.1 Bioinformatics analysis

[0029] EXPASY Compute pI / Mw (https: / / web.expasy.org / compute_pi / ), SMART (http: / / www.smart.embl-heidelberg.de / ), ESPript 3.0 (https: / / espript.ibcp.fr / ESPript / ESPript / ), SWISS-MODEL (https: / / www.swissmodel.expasy.org / ) and MEGA 11 were used to calculate the molecular weight, domain architecture, sequence conservation, three-dimensional structure and phylogenetic relationship of BmGPT (Bombyx mori GPT), respectively.

[0030] 1.2 Plasmid construction

[0031] The coding sequence of BmGPT (NCBI No. XP_004925546.1) was amplified using high-fidelity PCR, and the primer sequences are shown in SEQ ID NO.9-10 (Table 1). After separation by agarose gel, the recovered BmGPT amplification product was inserted into the pIZT-Myc vector using the ClonExpress II seamless cloning kit (Vazyme, Nanjing, China). The sequence of the recombinant construct was verified by Sanger sequencing.

[0032] 1.3 BmN cell line and transfection

[0033] Ovarian-derived BmN cells were cultured in TC-100 insect medium at 27°C, supplemented with 10% (v / v) fetal bovine serum (FBS), 100U / mL penicillin, and 0.1mg / mL streptomycin. For each well of a 6-well plate, 6μg of commercially synthesized small interfering RNA (siRNA) was mixed with 300μL serum-free TC-100, and 6μL RNAiMAX (Thermo Fisher Scientific) was mixed with another 300μL serum-free TC-100. The two parts were mixed, incubated at room temperature for 15 minutes, and then added to each well. The purified and endotoxin-free pIZT-BmGPT plasmid was transfected into BmN cells using a transfection reagent (NEOFECT). The siRNA sequences used in the present invention are shown in SEQ ID NO.11-14 (Table 1).

[0034] 1.4 BmNPV infection

[0035] BV-EGFP was obtained from the Key Laboratory of Sericulture Genetic Improvement, Jiangsu University of Science and Technology. Infection of BmNPV in BmN cell line: 10 μL BV-EGFP (1.0×10 8pfu / mL) were added to each well of a 6-well plate that had been transfected with siRNA or overexpression plasmid. At the indicated hours post-infection (h pi), green fluorescent protein (GFP) light emitted by each sample was detected using a Leica DMi8 fluorescence microscope. Cell pellets or supernatants containing BmNPV were then collected for experiments such as genomic DNA (gDNA) extraction, total RNA isolation, and reinfection of BmN cells.

[0036] Virus titer determination: To evaluate the proliferation of BmNPV at 24, 48, and 72 h post-infection, the cell supernatant at 48 h post-infection was collected and serially diluted 10-fold. Next, 10 μL of each dilution was added to 90 μL BmN cells (1.5 × 10 4 The cells were plated in 96-well plates (100 cells / well). Green fluorescence was recorded 72 hours after infection. The 50% tissue culture infectious dose (TCID50) was then calculated.

[0037] B. mori P50 silkworm larvae were reared on fresh mulberry leaves at 26 °C and 75% relative humidity. For in vivo BmNPV infection, 30 larvae at day 3 of the fifth instar were used for gene-specific knockdown. Each larva was injected with 3 μg of siRNA negative control (siNC) or siRNA Bombyx mori GPT (siBmGPT) and fed with 5 μL of ODVs (1 × 10 7 ODVs / mL). Midgut, hemocytes and fat bodies were collected 24, 48 and 72 hours after injection. VP39 expression and BmNPV copy number were measured by qPCR. Three biologically independent samples were used in the present invention. For tunicamycin (Tunicamycin, Catalog No. HY-A0098, dissolved in DMSO, MedChemExpress,) treatment, 5th instar larvae were orally fed with 1 μL tunicamycin (1 mM) and 1 μL DMSO.

[0038] 1.5 CCK8 cell activity assay

[0039] Before the cell viability assay, BmN cells transfected with siNC and siBmGPT were seeded in 96-well plates at a density of 4,000 cells per well and cultured for 12 hours. The proliferation rate of each group of cells was measured every 24 hours using a cell counting kit-8 (CCK-8, C0037, Beyotime). The optical density (OD) at 450 nm, which is linearly correlated with the number of viable cells, was measured using a SpectraMax i3 multi-mode microplate reader. 450 ).

[0040] 1.6 EdU cell proliferation assay

[0041] Using BeyoClick TM EdU incorporation assays of siRNA-transfected BmN cells were performed using the EDU-488 Cell Proliferation Kit (C0071S, Beyotime) according to the manufacturer's instructions. Briefly, 500 μL of EdU (10 μM) reagent was added to each well and incubated for 2 hours to label the cells. After washing three times with PBS, the cells were fixed in 4% formaldehyde solution for 15 minutes, permeabilized with 0.3% Triton X-100 for 15 minutes, and then incubated at room temperature in the dark for 30 minutes. The nuclei were stained using 1x Hoechst 33342 reagent, and the absorbance was measured at 488 nm using a SpectraMax i3 multi-mode microplate reader.

[0042] 1.7 TUNEL cell apoptosis assay

[0043] siRNA-transfected and BmNPV-infected BmN cells were tested using a terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) end labeling (TUNEL) assay kit (C1088; Beyotime,). Specifically: Cells were incubated with 4% formaldehyde for 30 minutes at room temperature for cell fixation. After rinsing three times with PBS, cells were incubated with PBS containing 0.3% Triton X-100 for 5 minutes at room temperature (cell permeabilization). Then, they were washed three times with PBS, incubated with a mixture of terminal deoxyribonucleotidyl transferase enzyme and Cy3-labeled dUTP, and incubated at 37°C in the dark for 1 hour (cell staining). Next, cell nuclei were stained with DAPI. Finally, images were captured using a fluorescence microscope, and the percentage of TUNEL-positive cells was quantified using ImageJ software.

[0044] 2.8 RNA extraction, reverse transcription (RT), gDNA extraction and quantitative real-time PCR (qPCR)

[0045] RNA was extracted using RNATRIzol reagent according to the manufacturer's instructions, and 2 μg of total RNA was reverse transcribed in a 20 μL reaction volume using All-in-One First-Strand Synthesis MasterMix. Each 6-well plate sample was homogenized in 1 mL of gDNA extraction buffer (10 mM Tris-HCl, 100 mM EDTA, 100 mM NaCl, 0.1 mg / mL proteinase K, 0.5% (m / V) SDS; pH = 8.0) and then incubated at 55°C / 12 hours for digestion. Next, an equal volume of 50% phenol-50% chloroform mixture was thoroughly mixed with the sample and centrifuged at 12000 rpm for 5 minutes to remove protein contamination. The supernatant was then mixed with two volumes of anhydrous ethanol and centrifuged at 12000 rpm for 5 minutes to precipitate gDNA. The samples were digested with RNaseA for 6 hours, extracted with phenol-chloroform, and ethanol precipitated. The purity and concentration of gDNA were determined using a NanoDrop 2000 spectrophotometer and agarose gel electrophoresis. Diluted cDNA and gDNA were used for qPCR analysis of VP39 and BmGPT on a Roche LC480 II real-time PCR thermal cycler. The qPCR master mix used was 2xAceQ Universal SYBR qPCR MasterMix. Three independent experiments were performed for each experiment, and three technical replicates were performed for each experiment. Relative expression levels were calculated using 2-ΔΔCt technology. B.mori RP49 (for RT-qPCR) and B.mori GAPDH (for gDNA-qPCR) were selected as internal reference genes. The primers used for RT-qPCR are shown in SEQ ID NO.1-8 (Table 1).

[0046] 1.9 Analysis of cell apoptosis by flow cytometry

[0047] The proportion of apoptotic cells was measured using the AnnexinV-FITC / PI apoptosis detection kit (C1062M, Beyotime). The collected cells were washed three times with PBS and resuspended in 100 μL of binding buffer. After that, the cell suspension was mixed with 5 μL of Annexin-VFITC and 10 μL of propidium iodide (PI) and incubated at room temperature for 15 minutes in the dark. Before flow cytometric analysis, 400 μL of binding buffer was added to the reaction mixture. The apoptotic cell rate was determined using flow cytometry.

[0048] 1.10 Inhibition of apoptosis based on reported concentration test on BmN cells

[0049] The indicated BmN cells were treated with Z-DEVD-FMK (T6005, Topscience; Shanghai, China) dissolved in DMSO at a working concentration of 10 μM to inhibit apoptosis. In the control group, an equal volume of DMSO was added to eliminate the potential effect of DMSO on the proliferation of BmNPV.

[0050] 1.11 Statistical analysis

[0051] Each experiment was performed in triplicate, and the data were analyzed using unpaired t-test and analysis of variance (ANOVA) using GraphPad Prism 9. The results are presented as mean ± standard deviation (SD). When an asterisk appears, the data are significantly different from the control group (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

[0052] Table 1: Sequences and their uses and numbers involved in the present invention

[0053]

[0054]

[0055] 2. Test Results

[0056] 2.1 Bioinformatics analysis results

[0057] The predicted BmGPT coding sequence (CDS) encodes a polypeptide containing 395 amino acid residues. The predicted molecular weight (Mw) and theoretical isoelectric point (pI) values ​​of the BmGPT protein are 44.78 kDa and 7.98, respectively. Sequence alignment using ESPript 3.0 software showed that there are 14 α-helices and 9 β-sheets in BmGPT, and these structures are largely conserved. Phylogenetic tree studies constructed using MEGA11 software and the traditional neighbor-joining method showed that BmGPT is closely related to Ostrinia nubilalis GPT.

[0058] 2.2 Spatiotemporal and inducible expression patterns of BmGPT

[0059] See also Figure 1 , in fifth instar day 3 larvae, the expression profile of BmGPT mRNA was identified in nine tissues by RT-qPCR; BmGPT mRNA was relatively uniformly expressed in these tissues ( Figure 1 In addition, the expression profile of BmGPT in eggs or developing midgut showed that BmGPT expression was higher in eggs ( Figure 1 In addition, the fifth instar larvae were infected by oral administration of ODV-type BmNPV virus, and the expression levels of BmGPT in the midgut, hemocytes and fat body were measured at 24, 48 and 72 hours after infection. Figure 1 As shown in Figures CE, BmNPV infection had no significant effect on the expression levels of BmGPT in these three tissues, except for a slight increase in BmGPT mRNA at 48 h post-infection. In addition, the expression of BmGPT in BmN cells infected with BmNPV at different time points showed that BmGPT expression increased from 2 to 36 h post-infection but remained limited. However, at 48 h post-infection, BmGPT levels showed a relatively low degree of reduction ( Figure 1 (F) These findings suggest that the expression level of BmGPT is relatively consistent across tissues, developmental stages, and viral stimulations, and that BmGPT may play an important role in BmNPV proliferation.

[0060] 3.3BmGPT regulates cell activity, proliferation and apoptosis

[0061] There are no reports on the biological function of the silkworm GPT gene. First, the full-length CDS of BmGPT was cloned into the pIZT-Myc vector and the construct was overexpressed in silkworm BmN cells. Cell activity was assessed using the CCK8 assay. Figure 2 As shown in Figure A, ectopic expression of BmGPT increased the activity of BmN cells. Conversely, knockdown of BmGPT reduced the activity of BmN cells ( Figure 2 In addition, knockdown of BmGPT reduced the proliferation of BmN cells ( Figure 2 C), and enhanced cell death by EdU incorporation and TUNEL staining, respectively ( Figure 2 Middle D). These findings suggest that BmGPT plays a key role in basic cellular functions through unknown mechanisms.

[0062] 3.4 BmGPT overexpression promotes the proliferation of BmNPV in BmN cells

[0063] In the present invention, the role of BmGPT in the proliferation of Bombyx mori nuclear polyhedrosis virus is focused on. Figure 3 As shown in Figure A, RT-qPCR data confirmed the successful overexpression of the BmGPT construct in BmN cells. Forced expression of BmGPT resulted in the expression of viral mRNA VP39 ( Figure 3 B) and BmNPV gDNA copy number ( Figure 3 In addition, images were taken to evaluate the green fluorescence signal in the BmN samples; the GFP intensity was significantly enhanced in cells overexpressing BmGPT compared to control cells ( Figure 3 In addition, the BmNPV titer was assessed using the TCID50 assay, and the results showed a significant increase in virus titer compared to the control group ( Figure 3These results indicate that BmGPT overexpression enhances the proliferation of BmNPV in BmN cells.

[0064] 3.5BmGPT knockdown inhibits the proliferation of BmNPV in BmN cells

[0065] Specific knockdown of BmGPT expression in BmN cells using siRNA. Figure 4 , the present invention successfully achieved knockdown of BmGPT using two different siRNAs, although the knockdown efficiencies in BmN cells were different ( Figure 4 (A). The proliferation of BmNPV was assessed using qPCR targeting viral VP39 mRNA and genomic DNA (gDNA) copies. Figure 4 As shown in Figures 4B and 4C, knockdown of BmGPT significantly reduced the proliferation of BmNPV in BmN cells. In addition, the green fluorescence signal in BmGPT knockdown cells was significantly reduced compared with that in siNC-treated cells ( Figure 4 At the same time, the BV titer was analyzed using the TCID50 assay. The results showed that the virus titer in BmGPT knockdown cells was significantly lower than that in control cells ( Figure 4 In conclusion, knockdown of BmGPT inhibited the proliferation of BmNPV in BmN cells.

[0066] Because knockdown of BmGPT promoted apoptosis in BmN cells, and apoptosis has been identified as an important mechanism against BmNPV, siNC and siBmGPT cells infected with BmNPV labeled with AnnexinV-FITC were evaluated using flow cytometry. Figure 5 As shown in Figures AB, treatment with BmGPT siRNA-1 and siRNA-2 resulted in a higher apoptosis rate in BmNPV-infected BmN cells than in siNC cells. To demonstrate that apoptosis is involved in the inhibition of BmNPV proliferation by BmGPT knockdown, siRNA-BmGPT-transfected BmN cells were treated with the caspase-3-specific inhibitor Z-DEVD-FMK. When apoptosis was blocked by Z-DEVD-FMK, viral VP39 ( Figure 6 A) level, gDNA copy number ( Figure 6 B) and the fluorescence intensity of BmNPV-EGFP virus was almost completely restored ( Figure 6 Middle C). The reason for the absence of a recovery effect at 24 h after infection may be that Z-DEVD-FMK requires longer than 24 h to exhibit an anti-apoptotic effect. Based on these findings, it can be expected that knockdown of BmGPT inhibits the proliferation of BmNPV at least in part by enhancing apoptosis of BmN cells.

[0067] 3.6 BmGPT inhibition reduces the proliferation of BmNPV in silkworms

[0068] siRNA was used to specifically knock down BmGPT expression in fifth instar larvae. Figure 7 , knockdown of BmGPT was achieved using two different siRNAs in the midgut, hemocytes, and fat body, respectively ( Figure 7 The proliferation of BmNPV was assessed by qPCR for viral VP39 mRNA and genomic DNA (gDNA) copies. Figure 7 DF and Figure 7 As shown in Figure GI, knockdown of BmGPT significantly reduced BmNPV proliferation in the midgut, hemocytes, and fat body. Tunicamycin is a commonly used GPT-specific inhibitor used to induce endoplasmic reticulum (ER) stress and unfolded protein response. Based on the aforementioned study of the role of BmGPT in BmN cells, we attempted to treat BmN cells with tunicamycin and detect the BmNPV gDNA copy number in BmN cells using VP39 and GP64-specific primers. The results are shown in Figure GI. Figure 8 As shown in A and B, tunicamycin significantly reduced the number of BmNPV gDNA copies in cells. Using the supernatant of BmN cells as a qPCR template, a significant reduction in BmNPV virus in the supernatant treated with tunicamycin was also detected ( Figure 8 In addition, immunoblotting experiments showed that the GP64 protein level was decreased in BmN cells treated with tunicamycin and their corresponding supernatants ( Figure 8 (E) Silkworms were also treated with tunicamycin to further verify the effect of BmGPT on the proliferation of BmNPV. Fig. 9 AC and Fig. 9 As shown in Figure DF, inhibition of BmGPT activity by tunicamycin significantly suppressed the levels of BmNPVVP39 mRNA and the copy number of BmNPV gDNA in the midgut, hemocytes, and fat body of silkworms. Taken together, these results indicate that inhibition of BmGPT by tunicamycin reduces the proliferation of BmNPV in vitro and in vivo and can play a protective role.

[0069] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.

Claims

1. The application of tunicamycin in the prevention and treatment of Bombyx mori nuclear polyhedrosis virus.

2. The application of tunicamycin in the preparation of preparations for the prevention and treatment of Bombyx mori nuclear polyhedrosis virus.

3. A preparation for preventing and treating Bombyx mori nuclear polyhedrosis virus, characterized in that: Tunicamycin is included in the formulation.

4. The preparation according to claim 3, characterized in that The preparation also contains a solvent required for dissolving tunicamycin. Preferably, the solvent includes one or more of glycerol, propylene glycol, aldehyde alcohol, and DMSO.

5. The preparation according to claim 3, characterized in that The tunicamycin content of the preparation is 1-10%.

6. The preparation according to claim 3, characterized in that The preparation was administered to silkworms by oral feeding, and the administration concentration was 0.01%.

7. The preparation according to claim 3, characterized in that The dosage form of the preparation is an emulsion.

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