Application of aspergillus versicolor exopolysaccharide AVP214-1 in preparation of medicine for resisting Singapore grouper iridovirus

Abstract

The invention discloses an application of aspergillus versicolor exopolysaccharide AVP214-1 in preparation of a medicine for resisting Singapore grouper iridovirus. It is found that in an in-vitro experiment, the AVP214-1 can significantly reduce the cytopathy degree induced by Singapore grouper iridovirus (SGIV), and the transcriptional level and the protein level of viral genes MCP and VP19 are reduced; furthermore, the AVP214-1 is applied to grouper in-vivo experiments, the AVP214-1 also has a good anti-SGIV effect, and a tissue slice result shows that an AVP214-1 treatment group relieves liver and spleen histopathologic changes caused by SGIV infection, and can significantly reduce the transcriptional level of viral genes MCP, VP19 and ICP18. The research provided by the invention has an extremely important significance in preventing or treating the fish iridovirus disease caused by the iridovirus of Singapore grouper by utilizing the AVP214-1.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. Specifically, it relates to the application of extracellular polysaccharide AVP214-1 of Aspergillus versicolor in the preparation of drugs against Singapore grouper iridovirus. Background Art

[0002] Grouper ( Epinephelus spp. ) is an important marine cultured fish in China. Because of its delicious meat, rich nutrition and extremely high edible value, it is deeply loved by people. In recent years, due to the increasing scale of aquaculture year by year, the continuous improvement of intensification degree and the outbreak of viral diseases caused by the deterioration of aquaculture environment have become the main reasons restricting the development of the industry. Among them, grouper iridovirus disease, especially the grouper iridovirus disease caused by Singapore grouper iridovirus (SGIV) of the genus Ranavirus, has caused a serious threat to the aquaculture industry in recent years due to its characteristics of fast transmission speed, strong pathogenicity and great difficulty in prevention and control, resulting in a significant increase in the mortality rate of aquaculture and an increasing economic loss year by year.

[0003] Patent CN116836821A discloses that Aspergillus versicolor SCAU214 is isolated from deep-sea sediment samples in the Mariana Trench (141°57'N, 10°51'E), fermented and cultured, its metabolites are extracted and purified, and its extracellular polysaccharide is obtained, named extracellular polysaccharide AVP214-1 of Aspergillus versicolor. Patent CN119868399A discloses the application of extracellular polysaccharide AVP141-A of Aspergillus versicolor in the preparation of drugs against grouper iridovirus, and AVP141-A can greatly reduce the degree of cytopathic effect caused by Singapore grouper iridovirus. However, the role of extracellular polysaccharide AVP214-1 of Aspergillus versicolor in grouper iridovirus has not been reported yet. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and provide an application of extracellular polysaccharide AVP214-1 of Aspergillus versicolor in the preparation of drugs against Singapore grouper iridovirus.

[0005] The second purpose of the present invention is to provide an application of extracellular polysaccharide AVP214-1 of Aspergillus versicolor in the preparation of drugs for preventing and / or treating fish iridovirus disease caused by Singapore grouper iridovirus infection.

[0006] The above-mentioned purpose of the present invention is achieved by the following technical solutions: Extracellular polysaccharide AVP214-1 of Aspergillus versicolor, with an average molecular weight of 8277 Da, and its structural formula is as follows: 。

[0007] The present invention has found through research that the extracellular polysaccharide AVP214-1 of Aspergillus versicolor has an inhibitory effect on Singapore grouper iridovirus. In in vitro experiments, AVP214-1 can significantly reduce the degree of cytopathic effect induced by Singapore grouper iridovirus. At the same time, it can also significantly reduce the transcriptional levels and protein levels of viral genes MCP and VP19, and this drug does not have toxic side effects on cells. Further, when AVP214-1 is used in in vivo experiments on groupers, AVP214-1 also has a good effect against Singapore grouper iridovirus. Compared with the control group, on the 3rd and 7th days after infection with SGIV, the liver and spleen of the group injected with AVP214-1 can significantly reduce the transcriptional levels of viral genes MCP, VP19, and ICP18. At the same time, the results of tissue section show that the AVP214-1 treatment group can alleviate the histopathological changes of the liver and spleen caused by SGIV infection, further verifying that AVP214-1 has an in vivo anti-SGIV effect, and this drug does not have toxic side effects on fish. This is of extremely important significance for using AVP214-1 to prevent or control Singapore grouper iridovirus.

[0008] Therefore, the present invention provides the application of the extracellular polysaccharide AVP214-1 of Aspergillus versicolor in the preparation of a drug for anti-Singapore grouper iridovirus.

[0009] The present invention also provides the application of the extracellular polysaccharide AVP214-1 of Aspergillus versicolor in the preparation of a drug for preventing and / or treating fish iridovirus disease caused by Singapore grouper iridovirus infection.

[0010] Further, the fish is a grouper.

[0011] Further, the drug achieves treatment by inhibiting the expression of grouper iridovirus genes MCP, VP19, and ICP18.

[0012] Further, the effective dose of the extracellular polysaccharide AVP214-1 in the drug is 0.5-45 mg / mL.

[0013] Even further, the effective dose of the extracellular polysaccharide AVP214-1 in the drug is 1-40 mg / mL.

[0014] Further, the drug also contains pharmaceutically acceptable excipients.

[0015] Even further, the excipients are selected from pharmaceutically acceptable carriers and excipients.

[0016] Further, the dosage form of the drug is selected from injection, powder, capsule, granule, etc.

[0017] Furthermore, the solvent of the injection is PBS buffer solution.

[0018] The research of the present invention shows that the extracellular polysaccharide AVP214-1 of Aspergillus versicolor can be diluted with a solvent and injected into fish bodies, providing a method for preventing and / or treating fish diseases caused by Singapore grouper iridovirus infection.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the application of extracellular polysaccharide AVP214-1 of Aspergillus versicolor in the preparation of drugs against Singapore grouper iridovirus. In vitro experiments show that AVP214-1 can significantly reduce the degree of cytopathic effect induced by Singapore grouper iridovirus, and can also significantly reduce the transcriptional level and protein level of viral genes MCP and VP19, and this drug will not produce toxic and side effects on cells. Further, when AVP214-1 is used in in vivo experiments on groupers, AVP214-1 also has a good effect against Singapore grouper iridovirus. Compared with the control group, the transcriptional levels of viral genes MCP, VP19, and ICP18 in the liver and spleen of the group injected with AVP214-1 can be significantly reduced on the 3rd and 7th days after infection with SGIV. At the same time, the results of tissue section show that the AVP214-1 treatment group can reduce the histopathological changes of the liver and spleen tissues caused by SGIV infection, further verifying that AVP214-1 has an in vivo anti-SGIV effect, and this drug will not produce toxic side effects on fish bodies. This is of extremely important significance for using AVP214-1 to prevent or control fish iridovirus disease caused by Singapore grouper iridovirus. Description of the Drawings

[0020] Figure 1 It is a graph showing the detection results of the cell viability of GS cells treated with different concentrations of AVP214-1 by CCK-8 experiment.

[0021] Figure 2 It is a graph showing the results of virus infection and replication levels in cells treated with different concentrations of AVP214-1.

[0022] Figure 3 It is a graph showing the detection results of the transcriptional level and protein level of viral genes in cells treated with different concentrations of AVP214-1.

[0023] Figure 4 It is a graph showing the survival rate results of groupers after injection of different concentrations of AVP214-1.

[0024] Figure 5 It is a graph showing the results of tissue sections of the liver and spleen of groupers after injection of different concentrations of AVP214-1.

[0025] Figure 6 It is a graph showing the survival rate of groupers within 14 days after being infected with SGIV.

[0026] Figure 7 It is a graph showing the results of tissue section of the liver and spleen of groupers on the 3rd day after being infected with SGIV.

[0027] Figure 8 It is a graph showing the transcriptional levels of viral genes in the liver and spleen of groupers on the 3rd and 7th days after being infected with SGIV. Detailed implementation manners

[0028] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0029] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0030] Example 1 Effect of extracellular polysaccharide AVP214-1 of Aspergillus versicolor on cell viability 1. The extracellular polysaccharide AVP214-1 of Aspergillus versicolor was prepared according to Patent CN116836821A.

[0031] 2. In order to detect the cytotoxic effect of different concentrations of AVP214-1 on grouper spleen (GS) cells, a Cell Counting Kit-8 (CCK-8) kit was used to detect the effect of different concentrations of AVP214-1 on the viability of the cultured cells. The GS cells were inoculated into a 96-well culture plate and cultured overnight in an incubator at 28 °C and 5% CO2. After the cells grew to confluence, 100 μL of fresh medium containing the corresponding concentration of AVP214-1 was added to each well. After treatment for 24 h, the cells were washed 3 times with fresh medium to remove the residual drug. Then, 100 μL of medium and 10 μL of Cell Counting Kit-8 (CCK-8, Uelandy) were added to each well, and the cells were incubated for 1-4 h in the dark. The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a multimode microplate reader (Thermo Fisher Science, USA) to reflect the cell metabolic activity.

[0032] The effect of different concentrations of AVP214-1 on the viability of grouper spleen (GS) cells after 24-hour treatment was evaluated by the CCK-8 experiment, and the results are as Figure 1As shown, compared with the untreated group, the cell viability of GS cells treated with AVP214-1 at concentrations of 0.5, 1, 3, 5, and 10 mg / mL did not change significantly. The above results indicate that AVP214-1 at concentrations of 0.5-10 mg / mL does not have a toxic effect on GS cells.

[0033] Example 2 In vitro antiviral activity experiment of Aspergillus versicolor extracellular polysaccharide AVP214-1 I. Experimental method 1. Preparation of SGIV: Transfer grouper spleen (GS) cells to a 25 cm 2 culture flask and culture them using Leibovitz's L15 medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS). The culture conditions are set at 28 °C and 5% CO2. After the cells adhere and grow to the logarithmic growth phase, inoculate Singapore grouper iridovirus (SGIV) at a multiplicity of infection (MOI = 0.5), and then continue to culture in a 28 °C incubator. Observe the cytopathic effect (CPE). After most cells show significant vacuolization, transfer the culture flask to an environment of -80 °C and perform 3 freeze-thaw cycles to fully lyse the cells and release virus particles. Finally, centrifuge to collect the virus supernatant, aliquot it, and store it in a -80 °C ultra-low temperature freezer for later use.

[0034] 2. Inoculate GS cells in a 24-well plate and culture overnight. After the cells grow to confluence, discard the original medium and add fresh medium containing different concentrations of AVP214-1 (0.5, 1, 3, 5, 10 mg / mL). Incubate AVP214-1 and GS cells in a 28 °C incubator for 2 hours, then inoculate Singapore grouper iridovirus (SGIV) at a multiplicity of infection (MOI) = 2, and continue to culture for 24 hours. Then, use an optical microscope to observe the cytopathic effect (CPE) and evaluate the inhibitory effect of different concentrations of AVP214-1 on virus infection.

[0035] 3. Fluorescent quantitative PCR method Extract total cellular RNA from cells in different groups and perform reverse transcription on the extracted total cellular RNA according to the reverse transcription system presented in Table 1 and the reverse transcription program shown in Table 2. The specific method is as follows: A. Extract cellular RNA using the Cell Total RNA Isolation Kit (Foregene, China). The specific steps are as follows: (1) Add 250 μL of Buffer cRL1 to the collected cell samples to fully lyse the cells.

[0036] (2) Transfer the fully lysed cell mixture to a DNA-Cleaning Column and centrifuge at 12,000 rpm (=13,400×g) for 2 minutes. Then remove the DNA-Cleaning Column and retain only the supernatant in the collection tube.

[0037] (3) Add Buffer cRL2 in a volume 1.6 times that of the supernatant obtained in step (2) to the supernatant, and then gently mix well.

[0038] (4) Transfer all of the above mixture to an RNA-only Column and centrifuge at 12,000 rpm (=13,400×g) for 1 minute. Discard the waste liquid in the collection tube.

[0039] (5) Add 500 μL of Buffer RW1 to the above RNA-only Column and centrifuge at 12,000 rpm (=13,400×g) for 1 minute. Discard the waste liquid in the collection tube.

[0040] (6) Add 700 μL of Buffer RW2 to the above RNA-only Column and centrifuge at 12,000 rpm (=13,400×g) for 1 minute. Discard the waste liquid in the collection tube.

[0041] (7) Repeat the operation in step (6) again.

[0042] (8) Centrifuge the above RNA-only Column with an empty tube at 12,000 rpm for 2 minutes.

[0043] (9) Transfer the purification column to a new EP tube, and drop 30 - 50 μL of pre-warmed RNase-Free ddH2O at 65°C onto the center of the purification column membrane, then let it stand at room temperature for 2 minutes.

[0044] (10) Place the EP tube containing the above purification column in a centrifuge at 12,000 rpm and centrifuge for 2 minutes to collect the RNA solution.

[0045] (11) Take out 1 μL of the RNA sample for concentration measurement, and take another 4 μL of the RNA sample for agarose gel electrophoresis detection to detect integrity.

[0046] (12) Finally, place the remaining RNA in a water bath at 65°C for 5 minutes for pre-denaturation, and then place it on ice after processing.

[0047] (13) Use ReverTra Ace qPCR RT Kit reverse transcriptase. After mixing the system evenly, place it in a PCR tube (operate according to the requirements in Table 1). Set the reverse transcription program on the PCR (set according to Table 2), and finally store it at 4°C or -20°C.

[0048] B. Use ReverTra Ace qPCR RT Kit (Toyobo, Japan) to reverse transcribe the total cellular RNA extracted to synthesize cDNA. The specific steps of the reverse transcription reaction are as follows: (1) Take 14 μL of RNA sample, place it in a water bath at 65°C for denaturation for 5 minutes, and then place it on ice for later use.

[0049] (2) Prepare the reaction system. Sequentially add 4 μL of 5×RT Buffer, 1 μL of Enzyme mix, 1 μL of Primer mix, and 14 μL of the previously prepared Total RNA.

[0050] (3) Place the prepared reaction system in a PCR instrument. The reaction program is 37°C for 15 minutes and 98°C for 5 minutes. After the reaction, store it at -80°C.

[0051] Table 1 Reverse transcription system

[0052] Table 2 Reverse transcription program

[0053] Then, according to the fluorescence quantitative system given in Table 3 and the fluorescence quantitative program shown in Table 4, use fluorescence quantitative PCR to detect the transcriptional levels of viral genes (MCP gene, VP19 gene) in different groups of cells respectively. The specific method steps are as follows: C. The operation steps of the fluorescence quantitative PCR experiment are as follows: Use 2×SYBR Green Real-time PCR Mix (Toyobo, Japan) to prepare the reaction system (Table 3), and use the β-Actin gene as an internal reference. Perform real-time fluorescence quantitative PCR reaction on a QuantStudio 5 real-time fluorescence quantitative PCR instrument according to the corresponding reaction program (Table 4), and finally use the 2 -ΔΔCT method to normalize the expression level of the target gene.

[0054] Table 3 Fluorescence quantitative system

[0055] Table 4 Fluorescence quantitative program

[0056] 4. Protein immunoblotting (Western blot) experiment (1) Protein sample preparation: Collect cell samples in a 12-well plate, add 40 μL of Pierce IP lysis buffer to lyse the cells. Add 10 μL of 5× protein loading buffer, and boil at 100 °C for 5 minutes to denature the protein. After centrifugation at 12,000×g for 3 minutes, take the supernatant for SDS-PAGE electrophoresis or store at -20 °C for later use. Take the supernatant for SDS-PAGE electrophoresis or store at -20 °C for later use after 3 minutes.

[0057] (2) SDS-PAGE gel preparation: Separating gel (10%): 5.0 mL of 30% acrylamide mixture, 2.6 mL of 1.5 M Tris-HCl (pH 8.8), 0.1 mL of 10% SDS, 0.1 mL of 10% ammonium persulfate, 0.01 mL of TEMED, make up to 10 mL with distilled water. After pouring, cover with isopropanol to isolate air and polymerize at room temperature for 30 - 60 minutes.

[0058] Stacking gel (5%): 0.6 mL of 30% acrylamide mixture, 0.5 mL of 1 M Tris-HCl (pH 6.8), 0.04 mL of 10% SDS, 0.04 mL of 10% ammonium persulfate, 0.01 mL of TEMED, make up to 4 mL with distilled water. After inserting the comb, let it stand at room temperature for 30 - 40 minutes.

[0059] (3) Electrophoresis and membrane transfer: After loading, perform two-stage electrophoresis, 70 V for 30 minutes in the stacking gel and 110 V for 60 minutes in the separating gel. After electrophoresis, activate the PVDF membrane with methanol and then perform protein transfer using the wet transfer method (100 mA, 60 minutes).

[0060] (4) Immunodetection: Block the transferred membrane with 5% non-fat milk at room temperature for 2 - 3 hours. After blocking, incubate with the primary antibody at room temperature for 2 - 3 hours or at 4 °C overnight. After the incubation of the primary antibody, wash 3 times with PBST (10 minutes each time). Incubate with the HRP-labeled secondary antibody at room temperature for 1 hour. After washing with PBST, use the HRP-DAB substrate color development kit (TIANGEN) and develop the color according to the operation instructions. Collect the image using a chemiluminescence imaging system and perform gray-scale analysis with Image J software.

[0061] II. Experimental results From Figure 2It can be seen that after treating cells with different concentrations of AVP214-1 (0.5, 1, 3, 5, 10 mg / mL) and infecting the cells with SGIV, compared with the control group, the degree of cytopathic effect induced by SGIV in the cells treated with AVP214-1 was significantly reduced after SGIV infection. The above results indicate that AVP214-1 has the effect of anti-SGIV.

[0062] It can be seen from Figure 3 that after treating cells with different concentrations (0.5, 1, 3, 5 mg / mL) of AVP214-1 and infecting the cells with SGIV, it was found that compared with the control group, after SGIV infection, the transcriptional level and protein level of the SGIV virus gene MCP in the cells treated with different concentrations (0.5, 1, 3, 5 mg / mL) of AVP214-1 both decreased significantly. At the same time, the transcriptional level of the SGIV virus gene VP19 also decreased significantly; and the inhibitory effect of AVP214-1 on virus gene expression showed an obvious dose-dependence, and its inhibitory effect enhanced with the increase of the AVP214-1 concentration.

[0063] Example 3 In vivo antiviral activity experiment of extracellular polysaccharide AVP214-1 from Aspergillus versicolor I. Experimental method 1. In the AVP214-1 safety concentration experiment, groupers were evenly divided into 5 groups, with 10 fish in each group, and were placed in 5 aquariums respectively. Four groups of fish were intraperitoneally injected with 100 μL of AVP214-1 solution containing different concentrations (10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL), and the control group was intraperitoneally injected with an equal amount of PBS. The health status of the fish was observed every day and the mortality was recorded. After 2 weeks, the liver and spleen were taken to prepare histological sections and observe pathological changes.

[0064] 2. In the antiviral activity assay, groupers were divided into 2 groups, with 40 fish in each group. One group was intraperitoneally injected with 100 μL of PBS containing 40 mg / mL AVP214-1, and the other group was intraperitoneally injected with an equal amount of PBS. After 7 days, both groups of fish were intraperitoneally injected with 100 μL of PBS containing 10 7.7 TCID 50 / mL SGIV. The health status of the fish was observed every day and the mortality was recorded until 14 days after infection. In addition, on the 3rd and 7th days after SGIV infection, the liver and spleen were obtained from the two groups of infected fish for detecting the transcription of virus genes or pro-inflammatory factors. At the same time, on the 3rd day after infection, the infected fish tissues (liver and spleen) were collected for histopathological examination.

[0065] II. Experimental method It can be seen from Figure 4 and Figure 5It can be seen that 14 days after injection of different concentrations of AVP214-1, groupers injected with 10 mg / mL, 20 mg / mL, 30 mg / mL, and 40 mg / mL AVP214-1 did not show any death, and there were no pathological changes in the livers and spleens of groupers injected with 40 mg / mL AVP214-1, indicating that intraperitoneal injection of 40 mg / mL AVP214-1 has no toxic effect on groupers.

[0066] It is known from Figure 6 that the groupers in the SGIV-infected group began to die on the 3rd day, and the cumulative mortality rate reached 50% by 14 days after infection. In contrast, the groupers in the AVP214-1 injection group began to die on the 4th day, and the cumulative mortality rate reached 26.7% by 14 days after infection, indicating that treatment with 40 mg / mL AVP214-1 can significantly reduce the mortality rate of fish caused by SGIV infection. The results of histopathological observation are as Figure 7 shown. Compared with the infected group treated with PBS, the pathological changes in the livers and spleens of the infected group treated with AVP214-1 were significantly alleviated. The research results show that treatment with AVP214-1 effectively alleviated the histopathological changes caused by SGIV infection. In addition, on the 3rd and 7th days after infection with SGIV, compared with the control group, the transcriptional levels of the SGIV virus genes MCP, VP19, and ICP18 in the livers and spleens of groupers in the AVP214-1 group were significantly decreased ( Figure 8 ).

[0067] The above specific embodiments provide a clearer and more complete description of the various technical features included in the technical solutions given by the present invention. It should be clear that the embodiments described herein are only a part of all the embodiments of the present invention, and do not cover all the embodiments. As long as these embodiments of the present invention are used, all other embodiments obtained by those skilled in the art without creative efforts are within the scope of protection of the present invention.

Claims

1. Use of extracellular polysaccharide AVP214-1 of Aspergillus versicolor in the preparation of a drug against Singapore grouper iridovirus.

2. Use of extracellular polysaccharide AVP214-1 of Aspergillus versicolor in the preparation of a drug for preventing and / or treating fish iridovirus disease caused by Singapore grouper iridovirus infection.

3. The application according to claim 2, characterized in that, The fish is a grouper.

4. The application according to any one of claims 1 or 2, characterized in that, The drug achieves treatment by inhibiting the expression of Singapore grouper iridovirus genes MCP, VP19, and ICP18.

5. The application according to any one of claims 1 or 2, characterized in that, The effective dose of extracellular polysaccharide AVP214-1 of Aspergillus versicolor in the drug is 0.5 - 45 mg / mL.

6. The application according to claim 5, characterized in that, The effective dose of extracellular polysaccharide AVP214-1 of Aspergillus versicolor in the drug is 1 - 40 mg / mL.

7. The application according to any one of claims 1 or 2, characterized in that, The drug also contains pharmaceutically acceptable excipients.

8. The application according to claim 7, wherein The excipients are selected from pharmaceutically acceptable carriers and excipients.

9. The application according to any one of claims 1 or 2, characterized in that, The preparation of the drug is an injection, powder, capsule, or granule.

10. The application according to claim 9, characterized in that The solvent of the injection is PBS buffer.

Images