Use of apolipoprotein III in prevention and treatment of nuclear polyhedrosis virus infection of bombyx mori

Abstract

The application discloses application of apolipoprotein III in prevention and treatment of Bombyx mori nuclear polyhedrosis virus infection and belongs to the cross field of molecular biology technology field and virus prevention and treatment. The application first finds that Bombyx mori apolipoprotein III (ApoLp-III) promotes biosynthesis and accumulation of ceramide, thereby inhibits mTORC1 signal pathway activity, induces cell G1 phase arrest, and effectively blocks a new mechanism of Bombyx mori nuclear polyhedrosis virus (BmNPV) replication. Based on the mechanism, the application provides application of ApoLp-III in inhibition of BmNPV proliferation, prevention and treatment of BmNPV infection, improvement of the ability of Bombyx mori to resist BmNPV or cultivation of a BmNPV-resistant Bombyx mori strain. The application provides a brand-new technical strategy and germplasm resource for realizing green, efficient and sustainable prevention and control of BmNPV.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of molecular biology and virus prevention and control, specifically involving the application of apolipoprotein III in the prevention and control of silkworm nucleopolyhedrovirus infection. Background Technology

[0002] Silkworm nucleopolyhedrovirus (BmNPV), a representative member of the genus Alpha baculovirus in the family Baculoviridae, is one of the most devastating diseases in sericulture. Silkworms infected with BmNPV exhibit typical symptoms, including decreased appetite, lethargy, abnormal body coloration (such as milky white or pale yellow spots on the body wall), and swelling of body segments, ultimately dying from organ failure. During the silkworm rearing season, an outbreak of BmNPV often leads to a sharp decline in cocoon yield and quality throughout the entire silkworm-producing area, sometimes even resulting in total crop failure and causing enormous economic losses to silkworm farmers. According to statistics, BmNPV causes hundreds of millions of US dollars in economic losses to the sericulture industry globally each year, seriously threatening the stability and sustainable development of the sericulture industry. Therefore, in-depth research into the pathogenic mechanism of BmNPV and the development of effective prevention and control strategies are of crucial strategic importance for ensuring the security and stability of the global silk supply chain.

[0003] For a long time, the control of BmNPV in sericulture has mainly relied on comprehensive preventive measures, including strict disinfection of silkworm rearing rooms and equipment, selection of high-quality virus-free silkworm eggs, strengthened feeding management, and the use of chemical or biological disinfectants. For example, the thorough disinfection of the silkworm rearing environment using chemical agents such as formaldehyde and bleaching powder is one of the effective means of controlling virus transmission. However, these chemical disinfectants not only pollute the environment, but long-term use may also pose a threat to the health of operators and easily induce drug-resistant variants of the virus. In terms of biological control, although some studies have explored the use of natural enemy microorganisms or plant extracts to inhibit BmNPV activity, their control effects are often unstable, and the mechanism of action is still unclear, making it difficult to promote and apply in large-scale production. In addition, traditional disease-resistant breeding methods, such as selecting resistant lines through multi-generation hybridization, have achieved certain results, but the breeding cycle is long, and the resistance level is easily affected by environmental factors, making it difficult to meet the urgent needs of modern sericulture for efficient, stable, and broad-spectrum resistance. The limitations of these existing prevention and control methods highlight the urgency and necessity of developing novel, efficient, and environmentally friendly antiviral strategies based on molecular biology and genetic engineering.

[0004] Apolipophorin III (ApoLp-III) is a soluble apolipoprotein widely found in insect hemolymph and is an important member of the apolipoprotein family. Its main physiological function is to participate in lipid transport and metabolism, playing an indispensable role in insect growth, development, energy supply, and reproduction. Whether ApoLp-III can inhibit the proliferation of silkworm virus has not yet been reported. Summary of the Invention

[0005] This invention discovers a novel mechanism by which silkworm apolipophorin-III (ApoLp-III) effectively blocks BmNPV replication by promoting the biosynthesis and accumulation of ceramides, thereby inhibiting mTORC1 signaling pathway activity and inducing G1 phase arrest. Based on the above findings, the purpose of this invention is to provide the application of silkworm apolipophorin-III in the prevention and control of silkworm nucleopolyhedrovirus infection. The amino acid sequence of the silkworm apolipophorin-III is shown in SEQ ID NO.1, and the coding sequence is shown in SEQ ID NO.2.

[0006] This invention discovered that the expression levels of ApoLp-III in the silk gland, midgut, and fat body tissues of silkworms were significantly upregulated 24-72 hours after infection with BmNPV. Overexpression of ApoLp-III in silkworm cells significantly inhibited BmNPV proliferation, while knockdown of ApoLp-III significantly promoted BmNPV proliferation. Lipidomics analysis revealed that in the stable cell line overexpressing ApoLp-III (pIZT-ApoLp-III), after BmNPV infection, the levels of various ceramide metabolites, including Cer d14:1 / d14:2 and Cer m34:2 / m37:0, were significantly higher than those in the control group; exogenous C6-ceramide effectively inhibited BmNPV replication in silkworm cells. Flow cytometry results showed that pIZT-ApoLp-III stable cells exhibited significant G1 phase arrest (approximately 90%), indicating that the cellular state necessary for viral DNA replication was effectively suppressed. Subsequent qRT-PCR analysis revealed a significant decrease in the mRNA level of RPTOR, a key component of the mTORC1 complex, in pIZT-ApoLp-III cells, leading to decreased mTORC1 activity. Furthermore, the transcription of key downstream effectors of mTORC1, including c-Myc, PCK2, and S6K1, was downregulated. These findings suggest that ApoLp-III-mediated cell cycle arrest is closely related to the inhibition of mTORC1 complex activity.

[0007] Treating stable cells overexpressing ApoLp-III with 50 nM of Myriocin, a specific inhibitor of ceramide synthase (SPT), for 24 h, followed by BmNPV infection, significantly reduced the antiviral effect of ApoLp-III. Furthermore, Myriocin treatment reversed the inhibitory effect of ApoLp-III on the mTORC1 complex. qRT-PCR analysis showed that the mRNA expression levels of RPTOR, a key gene of the mTORC1 complex, and downstream key genes c-Myc, PCK2, and S6K1 in Myriocin-treated cells were partially restored. These results demonstrate that ApoLp-III exerts its antiviral function by promoting the biosynthesis and accumulation of ceramide. The accumulated ceramide then inhibits the activation of the mTOR pathway, induces cell cycle arrest, and ultimately inhibits BmNPV replication.

[0008] This invention provides the application of silkworm apolipoprotein III, biological materials expressing silkworm apolipoprotein III, or ceramide in the in vitro inhibition of BmNPV proliferation. Preferably, the concentration of ceramide used to inhibit BmNPV proliferation in vitro is 10 μg / mL.

[0009] This invention also provides the application of silkworm apolipoprotein III, biological materials expressing silkworm apolipoprotein III, or ceramides in the preparation of products that inhibit BmNPV proliferation or prevent BmNPV infection. The products include reagents and pharmaceuticals.

[0010] This invention also provides the application of silkworm apolipoprotein III or biomaterials expressing silkworm apolipoprotein III in improving the resistance of silkworms to BmNPV. This application is achieved by overexpressing apolipoprotein III in silkworms.

[0011] This invention also provides the application of silkworm apolipoprotein III or biological materials expressing silkworm apolipoprotein III in the breeding of BmNPV-resistant silkworm strains. The BmNPV-resistant silkworm strain is a silkworm overexpressing apolipoprotein III.

[0012] The biomaterials expressing silkworm apolipoprotein III include any one of the following (1)-(3): (1) An expression cassette containing a nucleic acid molecule encoding silkworm apolipoprotein III; (2) A recombinant expression vector containing the expression cassette described in (1); (3) A host cell containing the expression cassette of (1) or a host cell containing the recombinant expression vector of (2).

[0013] The present invention also provides a method for improving the resistance of silkworms to BmNPV or for cultivating BmNPV-resistant silkworm strains, which includes: overexpressing apolipoprotein III in silkworms.

[0014] Advantages and beneficial effects of the present invention: This invention discovers ApoLp-III in silkworms as a novel host-guided antiviral target. Based on the "ceramide-mTOR" key signaling axis it regulates, novel biological agents such as recombinant proteins and small molecule agonists targeting this pathway can be developed. Furthermore, through gene editing and breeding technologies, transgenic antiviral silkworm lines that stably and highly express ApoLp-III can be cultivated, thus providing a new technical strategy and germplasm resource for achieving green, efficient, and sustainable control of BmNPV.

[0015] This invention not only provides a new perspective on elucidating the metabolic regulation mechanism of antiviral immunity in silkworms, but also provides important molecular targets and technical support for developing new strategies for the prevention and control of lepidopteran viral diseases that target host factors and have green and sustainable characteristics. Attached Figure Description

[0016] Figure 1 The changes in ApoLp-III transcription levels in different tissues of silkworms after BmNPV infection are shown. A: ApoLp-III expression in the silk gland; B: ApoLp-III expression in the midgut; C: ApoLp-III expression in the fat body.

[0017] Figure 2 The effect of ApoLp-III overexpression on BmNPV proliferation in silkworm cells. A: Overexpression efficiency; B: Change in BmNPV genome copy number after overexpression; C: TCID50 result after overexpression; D: VP39 protein expression level after overexpression.

[0018] Figure 3 The effect of ApoLp-III knockdown on BmNPV proliferation in silkworm cells. A: Knockdown efficiency; B: Change in BmNPV genome copy number after knockdown; C: TCID50 result after knockdown; D: VP39 protein expression level after knockdown.

[0019] Figure 4 Lipomic analysis results for ApoLp-III overexpressing stable cell lines. A: Multivariate statistical analysis (PLS-DA) results; B: Differential lipid clustering heatmap; C: VIP plot of upregulated ceramides identified.

[0020] Figure 5The effects of ApoLp-III overexpression on cell cycle arrest and transcriptional levels of mTOR pathway-related genes are shown. A: Scatter plot of forward and side scattering of pIZT cells; B: Scatter plot of forward and side scattering of pIZT-ApoLp-III cells; C: Cell cycle distribution of pIZT cells; D: Cell cycle distribution of pIZT-ApoLp-III cells; the vertical axis represents the relative number of cells; E: Statistical analysis of the proportion of pIZT and pIZT-ApoLp-III cells in G1 and S phases; F: Relative mRNA expression levels of c-Myc, PCK2, S6K1, and RPTOR.

[0021] Figure 6 The effect of exogenous C6-ceramide on BmNPV replication. A: C6-ceramide toxicity experiment on silkworm cells; B: Effect of different treatment groups on BmNPV genome copy number; C: Transcription levels of c-Myc, RPTOR, PCK2, and S6K1 genes in different treatment groups.

[0022] Figure 7 The effects of the ceramide synthase inhibitor Myriocin on the transcriptional levels of genes related to the BmNPV and mTOR signaling pathways. A: Toxicity of Myriocin in silkworm cells; B: Effects of different treatments on BmNPV genome copy number; CF: Transcriptional levels of c-Myc, RPTOR, PCK2, and S6K1 genes in different treatments. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that these embodiments are only some preferred technical solutions, and the scope of protection claimed by the present invention is not limited to the following embodiments.

[0024] The primer sequences used in the following examples are shown in Table 1 below.

[0025]

[0026] Example 1: Changes in ApoLp-III transcriptional levels in different tissues of silkworms after BmNPV infection Healthy P50 silkworm larvae, day 1 of the 5th instar, were randomly divided into an infection group (T) and a control group (C). The infection group was fed with a BmNPV virus suspension (1.0 × 10⁻⁶). 6Silkworms in both groups were fed mulberry leaves containing 1 BmNPV per mL, while the control group was treated with an equal volume of physiological saline. Midgut, silk gland, and fat body tissue samples were collected from both groups at 24 h, 48 h, 72 h, and 96 h post-infection. Each tissue sample from five silkworms constituted a biological replicate, with three biological replicates at each time point.

[0027] Total RNA was extracted from each sample using the Trizol method, and the RNA was reverse transcribed into cDNA using the TransScript® Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit. Real-time quantitative PCR (qRT-PCR) analysis was performed using the TransStart® Green qPCR SuperMix kit with ApoLp-III specific primers (sequences shown in Table 1), and the data were normalized using the silkworm BmGAPDH gene as an internal control. The qPCR reaction system is shown in Table 2 below.

[0028] Table 2

[0029] Amplification program: 94°C pre-denaturation for 30 seconds; followed by 40 cycles (94°C denaturation for 5 seconds, 60°C annealing / extension for 30 seconds). Experimental setup: 2 -ΔΔCT The relative expression levels of ApoLp-III were calculated using the method described above. Data are expressed as mean ± standard error (STDEV method), and t-tests were used to analyze the significance of differences between groups. Statistical significance was defined as p < 0.05. p<0.01 indicates a highly significant difference ( ).

[0030] result( Figure 1 The results showed that 24-48 h after silkworm infection with BmNPV, the expression level of ApoLp-III in the silk gland and midgut tissue was significantly upregulated; and 48-72 h after infection, the expression level of ApoLp-III in the fat body was significantly upregulated, thus confirming that BmNPV infection can induce the upregulation of ApoLp-III expression.

[0031] Example 2: Effects of ApoLp-III overexpression / knockdown on BmNPV proliferation in cells 1. Construction of overexpression vectors The pIZT-Mcherry-V5-His vector was linearized by double digestion with EcoRI and XhoI restriction endonucleases. Simultaneously, the ApoLp-III CDS sequence was amplified by PCR using silkworm cDNA as a template (primers were designed to contain EcoRI / XhoI restriction sites; sequences are shown in Table 1). After enzyme digestion and purification, the insert fragment was ligated to the linearized vector at an optimized molar ratio (1:3 to 1:7) using T4 DNA ligase. The ligation product was transformed into DH5α competent cells, plated on Zeocin-resistant plates, and positive clones were screened. The successful construction of the overexpression vector pIZT-ApoLp-III-Mcherry-V5-His was confirmed by colony PCR, double digestion verification, and sequencing (ensuring proper fusion of the reading frame with the V5 / His tag).

[0032] 2. siRNA synthesis siRNA synthesis was performed using in vitro transcription. First, interfering primers were designed and synthesized based on the ApoLp-III sequence (primer sequences are shown in Table 1), and annealing was used to form a double-stranded DNA template. The annealing program was: 95℃, 2 min; 95℃-22℃, 0.1℃ / sec; 22℃, 10 min. Subsequently, using the T7 RNAi Transcription Kit, the double-stranded template was mixed with NTP Mix, transcription buffer, and T7 enzyme, and in vitro transcription was performed at 37℃ for 4 h to obtain the initial transcription product. After transcription, a double-enzyme digestion system was prepared according to Table 3. DNase I and diluted RNase T1 were used to co-digest the reaction solution at 37℃ for 30 min to remove the DNA template and specifically cleave the transcription product, thereby obtaining siRNA of the desired size.

[0033] Table 3

[0034] Product purification was performed using the magnetic bead method. Room-temperature equilibrated RNA Clean Beads were mixed with the transcription digestion product and incubated at room temperature for 8-10 min to allow the siRNA to fully bind to the magnetic beads. The tube was placed on a magnetic rack to adsorb the magnetic beads until the solution became clear, and the supernatant was discarded. The magnetic beads were washed twice with pre-cooled 80% ethanol solution, briefly opened to dry, and then eluted with RNase-free H2O. The supernatant obtained by centrifugation was the purified siRNA. After concentration determination, it was stored at -80℃ for later use.

[0035] 3. Cell transfection Before transfection, select healthy BmN cells with a cell density of 65% at the bottom of the flask for whole-flask transfection; take 3 μg of overexpression vector pIZT-ApoLp-III-Mcherry-V5-His (empty vector pIZT-Mcherry-V5-His as a control) and 8 μL of Lipo8000™ transfection reagent, and add serum-free TC-100 medium to 250 μL and mix well; after replacing the old cell culture medium, add the plasmid-transfection reagent mixture, shake gently to distribute evenly, and incubate at 27℃ for 6-10 hours (whether to change the medium depends on the cell condition); observe fluorescence 36 hours after transfection to evaluate transfection efficiency.

[0036] 40 pmol of siRNA solution and 1.6 μL of Lipo8000™ transfection reagent were added sequentially to 50 μL of serum-free culture medium, gently mixed, and incubated at room temperature for 20 min. Then, this mixture was slowly added to the wells containing BmN cells, gently shaken, and cultured at 27°C.

[0037] 48 h after transfection, the old culture medium was discarded, and 1 μL of recombinant baculovirus (BmNPV-BV-eGFP) stock solution containing the eGFP gene was added at an inoculation amount of 3 with a multiplicity of infection (MOI) of 3 for infection. After 3 h of adsorption, the medium was replaced with fresh complete medium and cultured at 27 °C. Cells and supernatant were collected 48 h to 72 h after infection for absolute quantitative PCR, TCID50 viral titer and Western blot detection.

[0038] 4. Effect of ApoLp-III on BmNPV proliferation With BmNPV ie-1 The gene is a specific target (primer sequences are shown in Table 1). A standard curve was established using serial dilutions of positive standard plasmids with known copy numbers (linear range 1×10⁻⁶). 4 –1×10 8 The viral genome copy number in cell lysate was detected by absolute quantitative PCR (copy / μL).

[0039] Cell supernatant was collected simultaneously, and the viral titer was assessed using the TCID50 endpoint dilution method: the supernatant was serially diluted 10-fold and inoculated into BmN cells. After incubation at 27°C for 3-5 days, the infected wells showing green fluorescence (eGFP positive) were observed and counted under a fluorescence microscope. The half-maximal tissue culture infection dose (TCID50 / mL) was calculated according to the Reed-Muench formula.

[0040] In addition, silkworm cells were lysed using RIPA lysis buffer (Beyotime, China) to extract proteins. Equal volumes of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to a PVDF (Millipore) membrane. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with a primary antibody: rabbit polyclonal antibody VP39 (1:2000). Subsequently, the membrane was incubated with the corresponding horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) antibody (1:1000; Beyotime). The same sample was re-incubated with β-tubulin antibody (1:10000; TransGold) to verify consistency of loading. This allowed for a comprehensive evaluation of the dual inhibitory effect of ApoLp-III overexpression on viral replication and infectivity of mature viral particles.

[0041] The results showed that overexpression of ApoLp-III in silkworm cells significantly inhibited the proliferation of BmNPV. Figure 2 Knockdown of ApoLp-III significantly promotes the proliferation of BmNPV. Figure 3 ).

[0042] Example 3: Lipomic analysis results of ApoLp-III overexpressing stable cell lines 1. Construction of ApoLp-III stable cell lines The construction of stable cell lines first requires determining the optimal screening conditions: Untransfected BmN cells were treated with different concentrations of bleomycin using a kill curve assay. After culturing for 7-10 days, the lowest concentration that completely killed all cells within 10-14 days was selected as the optimal screening concentration. Subsequently, the experimental group plasmid pIZT-ApoLp-III-Mcherry-V5-His and the control group plasmid pIZT-Mcherry-V5-His were transfected into BmN cells using Lipo8000™ transfection reagent. Forty-eight hours after transfection, initial screening was performed using complete culture medium containing the optimal screening concentration of bleomycin. The screening medium was changed every 2-3 days to remove untransfected cells. After 2-4 weeks of continuous screening, when resistant cell colonies were observed to form and proliferate to a coverage rate of 30%-50% at the bottom of the culture vessel, the bleomycin concentration was reduced to a maintenance concentration for further expansion culture. By regularly monitoring the expression of mCherry red fluorescence using a fluorescence microscope, when the fluorescence positivity rate of the cell population stabilizes at 95%-100% after continuous passage, a stable cell line has been successfully obtained and can be cryopreserved to establish a cell bank.

[0043] 2. Lipid metabolomics experiments Control cells and ApoLp-III stabilized cells were infected with BmNPV at MOI=3. Cell samples were collected after 72 h and sent to a sequencing company for lipid metabolomics sequencing analysis. Three biological replicates were used for each group. Mass spectrometry analysis was performed using a QExactive instrument in ESI positive and negative ion mode. Lipid identification was performed using LipidSearch software with parameters set as follows: precursor and product ion tolerance of 5 ppm and product ion threshold of 5%. The absolute content was finally calculated by isotope internal standard method.

[0044] Differential metabolite analysis employed a combination of multivariate statistical and univariate tests for identification. First, partial least squares discriminant analysis (PLS-DA) was used to calculate variable importance projection values ​​(VIPs), and VIP ≥ 1 and T-test p-value < 0.05 were used as thresholds to screen for significantly differential metabolites. Then, z-score standardization and hierarchical clustering were performed on the differential metabolites, and heatmaps were generated to show their accumulation patterns in the sample and inter-group clustering trends, thereby comprehensively ensuring the reliability and biological relevance of the analysis results.

[0045] Studies have found that infection of ApoLp-III stable cells with BmNPV significantly induces the biosynthesis of ceramides Cer(d14:1_20:0)+HCOO, Cer(d14:2_20:0)+HCOO, Cer(m34:2+O)+HCOO, Cer(m37:0+O)+HCOO, and Cer(d12:0_23:0)+HCOO, with the expression levels of these metabolites upregulated by 1.7-4.0 times. Figure 4 ).

[0046] Example 4: ApoLp-III induces cell cycle arrest and inhibits mTORC1 complex activity The ApoLp-III stable cells (pIZT-ApoLp-III) constructed in Example 3 and control cells (pIZT) were seeded with BmNPV at a multiplicity of infection (MOI) of 3. After 48 h of infection, cells were collected, washed twice with phosphate-buffered saline (PBS), and fixed with pre-chilled anhydrous ethanol at -20°C for at least 1 h. After fixation, the ethanol was removed by centrifugation, and the cells were washed twice with PBS. Subsequently, the cells were resuspended in PBS, and RNase A (100 μg / mL) was added and treated at 37°C for 30 min. The cells were then filtered through a 40 μm cell sieve to obtain a single-cell suspension. The single-cell suspension was stained with 7-AAD (or propidium iodide, PI) at 4°C in the dark for 30–60 min. Cell cycle distribution was analyzed using a CytoFLEX flow cytometer (Beckman Coulter, USA), and the data were analyzed using ModFit LT software (Verity Software House). The results showed that pIZT-ApoLp-III cells exhibited significant G1 phase arrest, with up to 90% of cells arrested in this phase (p<0.01). Figure 5 (A-5E) indicates that the cellular state necessary for viral DNA replication was effectively suppressed.

[0047] Changes in the transcriptional levels of mTOR pathway-related genes (c-Myc, RPTOR, PCK2, S6K1; primers are shown in Table 1) in cells were detected using qPCR. Cells were collected 48 h after viral infection, and total RNA was extracted using the TRIzol method. The qPCR reaction system and procedure were as described in Example 1. -ΔΔC Relative gene expression levels were calculated using a qPCR method, with the pIZT group as the calibration sample and three biological replicates per group. qPCR results showed that the mRNA level of RPTOR, a key component of the mTORC1 complex, was significantly reduced in pIZT-ApoLp-III cells, leading to decreased mTORC1 activity. Consequently, the transcription of key downstream effectors of mTORC1, including c-Myc, PCK2, and S6K1, was downregulated. Figure 5 F).

[0048] Example 5: Adding exogenous C6-ceramide to silkworm cells inhibits BmNPV replication. To evaluate the effect of C6-ceramide on silkworm cell viability, the Cell Counting Kit-8 assay was used. Silkworm BmN cells were counted at 1×10⁻⁶. 4Cells were seeded per well in 96-well plates. After 12 h of adhesion, they were treated with different concentrations of C6-ceramide (dissolved in 10% DMSO + 90% corn oil) for 48 h, with an equal volume of solvent as a control. After treatment, the medium was replaced with fresh medium containing 10% CCK-8 reagent and incubated at 27°C for 2 h. Absorbance at 450 nm was measured using a microplate reader. Relative viability was calculated with the control group as 100% cell viability. Each group had 8 replicates, and the experiment was independently repeated 3 times. The results showed that C6-ceramide concentrations below 15 μg / mL had no significant effect on BmN cell viability. Figure 6 A).

[0049] BmN cells from silkworms in the logarithmic growth phase were seeded into cell culture plates. After cell attachment, the cells were divided into a control group and a treatment group. The control group had the same volume of solvent (10% DMSO + 90% Corn oil) added to the culture medium as the treatment group, while the treatment group had 10 μg / mL C6-ceramide added to the culture medium. After incubation at 27℃ for 48 h, both groups of cells were inoculated with BmNPV virus solution with a multiplicity of infection (MOI) of 3 and cultured for another 48 h after virus infection. Cell samples were collected from each group, and total DNA and RNA were extracted. The viral genome copy number was detected by absolute real-time PCR. The results showed that the viral genome copy number in the treatment group was significantly lower than that in the control group, indicating that exogenous C6-ceramide can effectively inhibit the replication of BmNPV in silkworm cells. Figure 6 B). The transcriptional levels of mTOR pathway-related genes were detected using relative quantitative PCR. The results showed that C6-ceramide treatment of BmN cells significantly downregulated the transcriptional levels of genes such as c-Myc, RPTOR, PCK2, and S6K1. Figure 6 C).

[0050] Example 6: Ceramide accumulation inhibits BmNPV replication by regulating mTORC1 complex activity. First, the effect on silkworm cell viability was detected using the CCK-8 assay to determine the safe working concentration of Myriocin, a specific inhibitor of ceramide synthase. BmN cells in the logarithmic growth phase were cultured at 1 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells / wells in 96-well plates. After 12 hours of culture, the cells adhered to the plates. Medium containing different concentrations of Myriocin was added to each well, with six replicates for each concentration. A blank control (cell-free medium) and a negative control (medium containing 0.1% DMSO) were also included. After 48 hours of further culture, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 27°C in the dark for 2 hours. The absorbance (OD) was then measured at 450 nm using a microplate reader. 450 Cell viability was calculated using the formula: Viability (%) = (Experimental group OD) / (Cell viability (%) 450- Blank Group OD 450 ) / (control group OD 450 - Blank group OD 450 The result showed that when the Myriocin concentration was ≤50 nM, the cell viability was not significantly different from that of the control group (P>0.05). Figure 7 (A) Therefore, 50 nM was chosen as the safe working concentration for subsequent experiments.

[0051] Four treatment groups were then set up: Group 1: empty vector cells + Myriocin + BmNPV (pIZT + Myriocin); Group 2: empty vector cells + DMSO + BmNPV (pIZT group); Group 3: ApoLp-III overexpression stable cells + DMSO + BmNPV (pIZT-ApoLp-III group); Group 4: ApoLp-III overexpression stable cells + Myriocin + BmNPV (pIZT-ApoLp-III + Myriocin group). After 24 hours of treatment, cells were inoculated with BmNPV virus solution at a multiplicity of infection (MOI) of 3. After 48 hours of culture, cells were collected to extract DNA, which was then used to inoculate the cells with BmNPV. ie-1 Using genes as targets, the viral genome copy number in each group was detected by absolute quantitative PCR. The results showed a significant difference in BmNPV genome copy number between group 4 and group 3, but both were significantly lower than those in group 2. Figure 7 B) indicates that Myriocin can partially restore BmNPV replication after inhibiting ceramide activity.

[0052] Changes in the transcriptional levels of mTOR pathway-related genes (c-Myc, RPTOR, PCK2, S6K1) in groups 1-4 were detected using qPCR. Cells were collected 48 hours after viral infection, and total RNA was extracted using the TRIzol method. The qPCR reaction system and procedure were as described in Example 1. -ΔΔC The relative gene expression levels were calculated using a method with the pIZT group as the calibration sample, and three biological replicates were set for each group. The results showed that the expression levels of related genes in group 4 were significantly higher than those in group 3, and the expression level of S6K1 recovered to a level close to that of control group 2. Figure 7 (C-7F) suggests that Myriocin blocks ceramide synthesis and can reverse the inhibitory effect of ApoLp-III on the mTOR signaling pathway.

[0053] The above embodiments are only used to help illustrate the present invention. The scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. Application of silkworm apolipoprotein III, biological materials expressing silkworm apolipoprotein III, or ceramide in the in vitro inhibition of BmNPV proliferation.

2. Application of silkworm apolipoprotein III, biological materials expressing silkworm apolipoprotein III, or ceramides in the preparation of products that inhibit BmNPV proliferation or prevent BmNPV infection.

3. The application according to claim 2, characterized in that: The products mentioned include reagents and pharmaceuticals.

4. Application of silkworm apolipoprotein III or biological materials expressing silkworm apolipoprotein III in improving the resistance of silkworms to BmNPV.

5. The application according to claim 4, characterized in that: The application was achieved by overexpressing apolipoprotein III in silkworms.

6. Application of silkworm apolipoprotein III or biological materials expressing silkworm apolipoprotein III in the breeding of BmNPV resistant silkworm strains.

7. The application according to claim 6, characterized in that: The BmNPV-resistant silkworm strain is a silkworm strain that overexpresses apolipoprotein III.

8. The application according to any one of claims 1-7, characterized in that: The biomaterials expressing silkworm apolipoprotein III include any one of the following (1)-(3): (1) An expression cassette containing a nucleic acid molecule encoding silkworm apolipoprotein III; (2) A recombinant expression vector containing the expression cassette described in (1); (3) A host cell containing the expression cassette of (1) or a host cell containing the recombinant expression vector of (2).

9. A method for improving the resistance of silkworms to BmNPV, characterized in that, include: Overexpression of apolipoprotein III in silkworms enhances their resistance to BmNPV.

10. A method for breeding BmNPV-resistant silkworm strains, characterized in that, include: Silkworms resistant to BmNPV were obtained by overexpressing apolipoprotein III in silkworms.

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