Application of aspergillus versicolor exopolysaccharide AVP141-A in preparation of medicine for resisting grouper iridovirus

Abstract

The invention discloses an application of aspergillus versicolor exopolysaccharide AVP141-A in preparation of a medicine for resisting grouper iridovirus. The research finds that the aspergillus versicolor exopolysaccharide AVP141-A has the effect of resisting the iridovirus of Singapore grouper, is relatively mild to cells, and does not bring toxic influence to the cells. Furthermore, the AVP141-A is used for preparing the medicine for resisting the grouper iridovirus, and the medicine has a remarkable antiviral effect: the degree of cytopathy caused by the Singapore grouper iridovirus can be greatly reduced, and meanwhile, the transcriptional level and the protein level of viral genes can be remarkably reduced; according to the present invention, the virus titer of the Singapore grouper iridovirus is significantly reduced, and the drug does not produce toxic side effects on cells, such that the important significance is provided for the resistance of the grouper iridovirus and the prevention and / or treatment of the fish diseases caused by the grouper iridovirus infection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular, relates to the application of Aspergillus versicolor extracellular polysaccharide AVP141-A in the preparation of drugs against grouper iridovirus. Background Art

[0002] Grouper ( Epinephelus spp. ) is an important marine farmed fish in my country with extremely high economic value. In recent years, due to the expansion of aquaculture scale year by year, the development of intensive and semi-intensive aquaculture models and the deterioration of the breeding environment, various infectious diseases, especially viral diseases, have spread widely, which are extremely harmful and difficult to prevent and control. Grouper iridovirus disease is a viral disease that has caused great harm to the grouper farming industry in recent years. The mortality rate of groupers has increased year by year, causing huge economic losses to the grouper farming industry.

[0003] Iridoviridae, also known as Icosahedral cytoplasmic deoxyriboviruses, is named iridovirus because the virus particles are arranged in an abnormally regular pattern at periodic intervals in the infected host or in the purified and concentrated virus precipitate, forming lattice planes that overlap each other, and appear blue or purple iridescent when illuminated by oblique light. At present, iridovirus is divided into 6 genera, among which those harmful to grouper include Lymphocystis disease virus (LCDV); Swelling virus genus: representative species include Infectious spleen and kidney necrosis virus (ISKNV), Red sea bream iridovirus (RSIV), etc.; Ranavirus genus: representative species include Epizootic haematopoietic necrosis virus (EHNV), Largemouth bass ranavirus (LMBV), Singapore grouper iridovirus (SGIV), etc.

[0004] In practical applications, aquatic drug control has the advantages of simple operation and a wide range of control. However, traditional chemical drug treatments have problems such as drug residues and environmental pollution. Therefore, finding natural, green and effective antiviral substances is of great significance to the development of grouper aquaculture.

[0005] Patent CN115260336A discloses an Aspergillus versicolor exopolysaccharide AVP141-A, which is isolated from a sample of Scleractinia in the South China Sea. Aspergillus versicolor The average molecular weight of the polysaccharide is 4082Da, which has an immunomodulatory effect on RAW264.7 macrophages. However, there is no report on the role of this polysaccharide in grouper iridovirus. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide the use of Aspergillus versicolor exopolysaccharide AVP141-A in the preparation of drugs against grouper iridovirus.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions: The present invention has found that the Aspergillus versicolor exopolysaccharide AVP141-A has the effect of resisting Singapore grouper iridovirus, and it is relatively mild to cells and will not cause toxic effects on cells. AVP141-A at a concentration of 1 to 5 mg / ml will not cause toxic effects on cells. Therefore, the present invention applies AVP141-A to the preparation of anti-grouper iridovirus drugs, and the prepared drug has a significant anti-grouper iridovirus effect: it can greatly reduce the degree of cell pathological changes caused by Singapore grouper iridovirus, and at the same time, it can also significantly reduce the transcription level of viral genes (MCP gene, ICP 18 gene, VP 19 gene) and protein (MCP protein) level, significantly reduce the virus titer of Singapore grouper iridovirus, and the drug will not produce toxic side effects on cells, which is of great significance for resisting grouper iridovirus and preventing and / or treating diseases caused by grouper iridovirus infection in fish.

[0008] Therefore, the present invention first provides the use of Aspergillus versicolor extracellular polysaccharide AVP141-A in preparing drugs against grouper iridovirus.

[0009] The present invention also provides the use of Aspergillus versicolor extracellular polysaccharide AVP141-A in preparing medicines for preventing and / or treating fish diseases caused by grouper iridovirus infection.

[0010] Furthermore, the grouper iridovirus is Singapore grouper iridovirus (SGIV).

[0011] Furthermore, the fish is grouper.

[0012] Furthermore, the disease is iridovirus disease.

[0013] Furthermore, the effective dosage of Aspergillus versicolor extracellular polysaccharide AVP141-A in the drug is 1 to 5 mg / mL.

[0014] Preferably, the effective dose of Aspergillus versicolor exopolysaccharide AVP141-A in the drug is 5 mg / mL.

[0015] Furthermore, the medicine also contains pharmaceutically acceptable excipients.

[0016] Preferably, the auxiliary material is selected from at least one of a pharmaceutically acceptable carrier, an excipient or a solvent.

[0017] Furthermore, the dosage form of the drug is selected from at least one of tablets, capsules, powders, granules, pills or solutions.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention first provides the use of Aspergillus versicolor exopolysaccharide AVP141-A in the preparation of drugs against grouper iridovirus. The present invention has found that Aspergillus versicolor exopolysaccharide AVP141-A has the effect of resisting Singapore grouper iridovirus, and it is relatively mild to cells and will not cause toxic effects on cells. AVP141-A at a concentration of 1 to 5 mg / ml will not cause toxic effects on cells. AVP141-A is further used to prepare drugs against grouper iridovirus, and the drug has achieved significant antiviral effects: it can greatly reduce the degree of cell pathological changes caused by Singapore grouper iridovirus, and at the same time, it can also significantly reduce the transcription level of viral genes (MCP gene, ICP 18 gene, VP 19 gene) and protein (MCP protein) level, significantly reduce the virus titer of Singapore grouper iridovirus, and the drug will not have toxic side effects on cells, which is of great significance for resisting grouper iridovirus and preventing and / or treating fish diseases caused by grouper iridovirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the results of cell viability detection of GS cells treated with different concentrations of AVP141-A using CCK-8 experiment.

[0020] Figure 2 The graph shows the degree of viral infection and replication in cells after the cells were treated with different AVP141-A concentrations.

[0021] Figure 3 The results of the detection of the transcription level (A) and protein level (B) of viral genes in cells after the cells were treated with different AVP141-A concentrations.

[0022] Figure 4The graph shows the degree of viral infection and replication in cells treated with AVP141-A 12h and 24h after infection with SGIV compared with the control group.

[0023] Figure 5 The graph shows the results of the detection of viral gene transcription level (A), protein level (B) and titer (C) in different cells treated with AVP141-A 12h and 24h after SGIV infection compared with the control group. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0025] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0026] Example 1 Aspergillus versicolor extracellular polysaccharide AVP141-A was prepared according to patent CN115260336A.

[0027] Preparation of SGIV: Grouper spleen (GS) cells were transferred to 25 cm² culture flasks and grown in Leibovitz's L15 medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum at 28°C. When the cells were attached and entered the logarithmic growth phase, Singapore grouper iridovirus (SGIV) was added to the culture system at a multiplicity of infection (MOI) of 0.5, and then placed in an incubator for culture. When most cells showed obvious vacuolation, the culture flask was placed at -80°C and repeatedly frozen and thawed three times to release the SGIV virus completely. The culture medium was then aliquoted and stored at -80°C.

[0028] In order to detect the toxic effects of different concentrations of AVP141-A on GS cells, the Cell Counting Kit-8 (CCK-8) kit was used to detect the effects of different concentrations of AVP141-A on the viability of cultured cells. GS cells were seeded in a 96-well plate and cultured overnight at 28°C. When the cells grew into a monolayer, the corresponding concentration of AVP141-A was added and the plate was returned to 28°C and 5% CO. 2After 24 h of treatment, the cells were washed three times with fresh culture medium, and 100 μL of culture medium and 10 μL of Cell Counting Kit-8 (CCK-8, Uelandy) were added to each well. The cells were cultured in a 28 °C incubator for 1 to 4 h. The absorbance was measured at 450 nm using a multi-mode microplate reader (Thermo Fisher Science, USA).

[0029] Depend on Figure 1 It can be seen that the cell viability of GS cells cultured in LeWBovitz's L15 medium containing different concentrations of AVP141-A was detected using CCK-8 experiment. The results showed that the cell viability of GS cells treated with AVP141-A at concentrations of 1, 3, and 5 mg / mL for 24 hours did not change significantly compared with the group not treated with AVP141-A. The above results indicate that AVP141-A at a concentration of 1 to 5 mg / mL will not have a toxic effect on cells.

[0030] Example 2 AVP141-A antiviral activity experiment GS cells were seeded in a 24-well plate and cultured overnight at 28°C. When the cells grew into a monolayer, AVP141-A at a concentration that was non-toxic to the cells was added. AVP141-A and GS cells were incubated in a 28°C incubator for 2 hours, and then SGIV (MOI = 2) was added. After 24 hours, the pathological changes of GS cells after adding different concentrations of AVP141-A were observed. Total cell RNA was then extracted from cells in different groups, and the extracted total cell RNA was reverse transcribed according to the reverse transcription system presented in Table 1 and the reverse transcription procedure shown in Table 2. The specific method is as follows: 1. Extract cellular RNA using the Cell Total RNA Isolation Kit (Foregene, China). The specific steps are as follows: (1) Add 250 μL Buffer cRL1 to the collected cell sample.

[0031] (2) Transfer the lysed cell mixture to the DNA-Cleaning Column and centrifuge at 12,000 rpm (= 13,400 × g) for 2 minutes. Then remove the DNA-Cleaning Column and keep only the supernatant in the collection tube.

[0032] (3) Add 1.6 times the volume of Buffer cRL2 to the supernatant obtained in step (2) and mix gently.

[0033] (4) Transfer the entire mixture to the RNA-only Column and centrifuge at 12,000 rpm (= 13,400 × g) for 1 minute. Discard the waste liquid in the collection tube.

[0034] (5) Add 500 μL of Buffer RW1 to the purification column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.

[0035] (6) Add 700 μL of Buffer RW2 to the purification column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.

[0036] (7) Repeat step (6) again.

[0037] (8) Centrifuge the collection tube containing the purification column at 12,000 rpm for 2 minutes.

[0038] (9) Transfer the purification column to a new EP tube and drip 30-50 μL of RNase-Free ddH2O preheated at 65°C toward the center of the purification column membrane. 2 O, and then leave at room temperature for 2 minutes.

[0039] (10) Centrifuge the EP tube containing the purification column at 12,000 rpm for 2 minutes to collect the RNA solution.

[0040] (11) Take out 4 μL of RNA sample for agarose gel electrophoresis and take another 1 μL of sample for concentration determination.

[0041] (12) Finally, place the remaining RNA in a 65°C water bath for 5 minutes for pre-denaturation and place on ice after treatment.

[0042] (13) Use the ReverTra Ace qPCR RT Kit reverse transcriptase, mix the system evenly and place it in a PCR tube (follow the requirements in Table 1), set the reverse transcription program on the PCR (follow the requirements in Table 2), and finally store it at 4°C or -20°C.

[0043] 2. Use the ReverTra Ace qPCR RT Kit (Toyobo, Japan) to reverse transcribe the extracted total cell RNA to synthesize cDNA. The reverse transcription reaction steps are as follows: (1) Take 14 μL of RNA sample and place it in a 65°C water bath for denaturation for 5 minutes, then place it on ice for later use.

[0044] (2) Prepare the reaction system by adding 4 μL of 5×RT Buffer, 1 μL of Enzyme mix, 1 μL of Primer mix, and 14 μL of Total RNA prepared previously.

[0045] (3) Place the prepared reaction system into the PCR instrument. The reaction program is 37°C for 15 minutes and 98°C for 5 minutes. After the reaction is completed, store it at -80°C.

[0046] Table 1 Reverse transcription system

[0047] Table 2 Reverse transcription procedures

[0048] Next, according to the fluorescence quantitative system given in Table 3 and the fluorescence quantitative procedure shown in Table 4, the transcription level and protein level of viral genes (MCP gene, ICP 18 gene, VP 19 gene) in different groups of cells were detected by fluorescence quantitative PCR and protein immunoblotting (Western blot) experiments. The specific method steps are as follows: 1. 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 β-Actin gene as the internal reference, and perform real-time fluorescence quantitative PCR reaction on QuantStudio 5 real-time fluorescence quantitative PCR instrument according to the corresponding reaction program (Table 4). -ΔΔCT Methods The expression levels of target genes were normalized.

[0049] Table 3 Fluorescence quantification system

[0050] Table 4 Fluorescence quantification program

[0051] 2. The specific steps of the Western blot experiment are as follows: (1) Sample preparation: Collect cell samples in a 12-well plate and add 40 μL Pierce IP lysis buffer to lyse the cells. Add 10 μL 5× protein loading buffer, boil in boiling water for 5 minutes to denature the protein, centrifuge at 12,000×g for 3 minutes, and take the supernatant for electrophoresis or store at -20°C for later use; (2) Glue preparation: Pour the lower layer of separation gel between clean glass plates. The composition includes 30% acrylamide-bisacrylamide mixture (5.0 mL), 1.5M Tris-HCl (pH 8.8) (2.6 mL), 10% SDS (0.1 mL), 10% ammonium persulfate (0.1 mL) and TEMED (0.01 mL). Use distilled water to make the volume 10 mL. After pouring quickly, carefully add a layer of isopropanol on the gel surface to prevent the gel surface from contacting with air and oxidizing. After the separation gel solidifies (about 30 to 60 minutes), pour off the upper layer of liquid, absorb the remaining liquid with filter paper, and then pour the upper layer of concentrated gel. The stacking gel formula is 30% acrylamide-bisacrylamide mixture (0.6 mL), 1M Tris-HCl (pH 6.8) (0.5 mL), 10% SDS (0.04 mL), 10% ammonium persulfate (0.04 mL) and TEMED (0.01 mL), and the volume is adjusted to 4 mL with distilled water. Insert the comb and wait for the stacking gel to solidify (about 30 to 40 minutes).

[0052] (3) Sample addition: Carefully remove the comb, place the gel in the electrophoresis tank, and add 1× SDS-PAGE electrophoresis buffer. Slowly add an equal mass of the protein sample to be tested into the sample well. At the same time, add a protein marker of appropriate size to the adjacent well to determine the molecular weight of the target protein.

[0053] (4) Electrophoresis: Run the upper gel at 70 V for 30 min and the lower gel at 110 V for 60 min, until the bromophenol blue indicator reaches the bottom of the gel.

[0054] (5) Transfer: Soak the PVDF membrane (Millipore) in methanol for 1-2 minutes to activate it. After the electrophoresis, remove the protein gel and assemble the transfer sandwich structure in the order of "sponge-filter paper-gel-membrane-filter paper-sponge". Use a glass rod or roller to gently roll out the bubbles. Place the transfer sandwich in the transfer device and use the wet transfer method at a constant current of 100 mA for 60 minutes to transfer the protein blot in the protein gel to the PVDF membrane.

[0055] (6) Blocking: Place the transferred membrane in 5% skim milk prepared with PBST and incubate with slow shaking at room temperature for 2 to 3 hours, or overnight at 4°C.

[0056] (7) Primary antibody incubation: Remove the blocked membrane from the blocking solution and place it in the primary antibody solution diluted with the blocking solution. Incubate slowly with shaking at room temperature for 2 to 3 hours, or overnight at 4°C.

[0057] (8) Secondary antibody incubation: After the primary antibody incubation is completed, place the membrane in PBST buffer and wash it slowly with shaking at room temperature for 10 minutes each time, for a total of 3 washes. Place the washed membrane in the secondary antibody solution diluted with blocking buffer and incubate it slowly with shaking at room temperature for 1 hour.

[0058] (9) Color development: Wash the membrane three times with PBST buffer for 10 minutes each time to remove unbound secondary antibody. Use the enhanced HRP-DAB substrate color development kit (TIANGEN) and follow the instructions for color development.

[0059] (10) Photography and analysis: Take photos using a chemiluminescence imaging system and perform grayscale analysis on the images using Image J software.

[0060] The results of the intracellular viral infection and replication after cells were treated with different AVP141-A concentrations can be referred to Figure 2 After treating cells with different concentrations of AVP141-A, the results of the detection of viral gene transcription and protein levels in cells can be viewed Figure 3 .

[0061] Depend on Figure 2 It can be seen that after treating cells with AVP141-A at different concentrations (1, 3, and 5 mg / mL) and infecting cells with SGIV, the degree of SGIV-induced cytopathic effect was significantly reduced after AVP141-A treatment. The above results indicate that AVP141-A has anti-SGIV efficacy.

[0062] Depend on Figure 3 It can be seen that after treating cells with different concentrations of AVP141-A (1, 3, 5 mg / mL) and infecting cells with SGIV, the results showed that under the conditions of AVP141-A treatment at concentrations of 1, 3, and 5 mg / mL, the transcription level and protein level of the MCP gene of the SGIV virus were significantly decreased. At the same time, the transcription levels of the ICP 18 gene and VP 19 gene in the SGIV virus also decreased significantly; and the inhibitory effect of AVP141-A on the transcription and protein expression of viral genes in cells was concentration-dependent. The above results show that AVP141-A has significant anti-SGIV activity.

[0063] Example 3 GS cells were treated with AVP141-A at a concentration of 5 mg / mL, and the effect of SGIV infection on the anti-SGIV activity of the cells at different times was studied. Specifically, GS cells were inoculated in a 12-well plate and cultured overnight at 28°C. When the cells were fully grown in a monolayer, AVP141-A at a concentration of 5 mg / mL was added, and AVP141-A and GS cells were incubated in a 28°C incubator for 2 hours. After that, SGIV (MOI=2) was added, and the pathological changes of GS cells co-incubated with AVP141-A were observed after 12h and 24h compared with the control group. Protein samples or RNA samples were collected, and the transcription level of the MCP gene and the expression level of the protein were detected.

[0064] At the same time, the cell samples and supernatant obtained from the above experimental groups were collected in the same centrifuge tube, placed at -80°C and repeatedly frozen and thawed three times, and the virus titer in the cells was detected. The specific operation steps are as follows: (1) Subculture the GS cells in good condition into a 96-well cell culture plate and culture overnight; (2) After the cells evenly cover 80-90% of the bottom of the cell plate, take out the virus-infected sample that has been frozen and thawed, shake and mix after thawing, and dilute it 10 times with serum-free culture medium. Add 100 μL of the gradient diluted sample to each well of the cell plate; (3) Dilute 10 1 ~10 11 The virus was added to a 96-well plate, 100 μL per well, and a normal control group without virus infection was set up, with 8 parallels in each group; (4) Observe the CPE caused by the virus every day and record and calculate the TCID of the sample according to the Reed-Muench method 50 .

[0065] The results are as follows Figure 4 , Figure 5 As shown, the effect of AVP141-A in inhibiting the transcription of MCP gene and VP 19 gene of SGIV virus increases with the extension of SGIV infection time, and the effect of reducing SGIV virus titer also increases with the extension of infection time, indicating that with the extension of SGIV infection time, the activity of AVP141-A in inhibiting SGIV infection gradually increases.

Claims

1. Application of Aspergillus versicolor extracellular polysaccharide AVP141-A in the preparation of drugs against grouper iridovirus.

2. Application of Aspergillus versicolor exopolysaccharide AVP141-A in the preparation of drugs for preventing and / or treating fish diseases caused by grouper iridovirus infection.

3. The use according to claim 1 or 2, characterized in that: The grouper iridovirus is Singapore grouper iridovirus.

4. The application according to claim 2, characterized in that: The fish is grouper.

5. The application according to claim 2, characterized in that: The disease is iridovirus disease.

6. The use according to claim 1 or 2, characterized in that: The effective dosage of Aspergillus versicolor extracellular polysaccharide AVP141-A in the medicine is 1-5 mg / mL.

7. The use according to claim 6, characterized in that: The effective dosage of Aspergillus versicolor extracellular polysaccharide AVP141-A in the drug is 5 mg / mL.

8. The use according to claim 1 or 2, characterized in that: The medicine also contains pharmaceutically acceptable excipients.

9. The use according to claim 8, characterized in that: The auxiliary material is selected from at least one of a pharmaceutically acceptable carrier, an excipient or a solvent.

10. The use according to claim 1 or 2, characterized in that: The dosage form of the drug is selected from at least one of tablets, capsules, powders, granules, pills or solutions.

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