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

Abstract

The present invention discloses the application of Aspergillus versicolor extracellular polysaccharide AVP141-A in the preparation of drugs against grouper iridovirus. The present invention studies and discovers that Aspergillus versicolor extracellular polysaccharide AVP141-A has the effect of resisting Singapore grouper iridovirus, and it is relatively mild to cells and will not cause toxic effects to cells. Further, AVP141-A is used in the preparation of drugs against grouper iridovirus, and the said drugs have achieved remarkable antiviral effects: it can greatly reduce the degree of cytopathic effect caused by Singapore grouper iridovirus, and at the same time can significantly reduce the transcriptional level and protein level of viral genes, significantly reduce the virus titer of Singapore grouper iridovirus, and moreover, the said drugs will not produce 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.

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 extracellular polysaccharide AVP141-A of Aspergillus versicolor, which is isolated from a sample of stony coral (Scleractinia) in the South China Sea and obtained by fermenting and re-isolating the strain of Aspergillus versicolor ( Aspergillus versicolor SCAU141). Its average molecular weight is 4082 Da, and it has an immunomodulatory effect on RAW264.7 macrophages. However, there is no report on the role of this polysaccharide in grouper iridovirus yet. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art, and provide the application of extracellular polysaccharide AVP141-A of Aspergillus versicolor in the preparation of drugs against grouper iridovirus.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] The present invention discovers through research that extracellular polysaccharide AVP141-A of Aspergillus versicolor has the effect of resisting Singapore grouper iridovirus, and it is relatively gentle to cells and will not cause toxic effects to cells. AVP141-A with a concentration of 1-5 mg / ml will not produce toxic effects on cells. Therefore, the present invention applies AVP141-A to the preparation of drugs against grouper iridovirus, and the prepared drug has a significant effect against grouper iridovirus: it can greatly reduce the degree of cytopathic effect caused by Singapore grouper iridovirus, and at the same time can significantly reduce the transcriptional level and protein (MCP protein) level of virus genes (MCP gene, ICP 18 gene, VP 19 gene), significantly reduce the virus titer of Singapore grouper iridovirus, and this 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 infecting fish.

[0009] Therefore, the present invention first provides the application of extracellular polysaccharide AVP141-A of Aspergillus versicolor in the preparation of drugs against grouper iridovirus.

[0010] The present invention also provides the application of extracellular polysaccharide AVP141-A of Aspergillus versicolor in the preparation of drugs for preventing and / or treating fish diseases caused by grouper iridovirus infection.

[0011] Further, the grouper iridovirus is Singapore grouper iridovirus (SGIV).

[0012] Further, the fish is grouper.

[0013] Further, the disease is iridovirus disease.

[0014] Furthermore, the effective dose of Aspergillus versicolor extracellular polysaccharide AVP141-A in the drug is 1-5 mg / mL.

[0015] Preferably, the effective dose of Aspergillus versicolor extracellular polysaccharide AVP141-A in the drug is 5 mg / mL.

[0016] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0017] Preferably, the excipients are selected from at least one of pharmaceutically acceptable carriers, excipients or solvents.

[0018] Furthermore, the dosage form of the drug is selected from at least one of tablets, capsules, powders, granules, pills or solutions.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention first provides the application of Aspergillus versicolor extracellular polysaccharide AVP141-A in the preparation of drugs against grouper iridovirus. The present invention finds that Aspergillus versicolor extracellular polysaccharide AVP141-A has the effect of resisting Singapore grouper iridovirus, and it is relatively mild to cells and will not cause toxic effects to cells. AVP141-A with a concentration of 1-5 mg / ml will not produce toxic effects on cells. Further, AVP141-A is used in the preparation of drugs against grouper iridovirus, and the drug has achieved remarkable antiviral effects: it can greatly reduce the degree of cytopathic effect caused by Singapore grouper iridovirus, and at the same time can significantly reduce the transcriptional level and protein (MCP protein) level of virus genes (MCP gene, ICP 18 gene, VP 19 gene), significantly reduce the virus titer of Singapore grouper iridovirus, and this drug will not produce toxic side effects on cells, which is of great significance for anti-grouper iridovirus and the prevention and / or treatment of fish diseases caused by grouper iridovirus infection. Description of the Drawings

[0021] Figure 1 It is a result diagram of detecting the cell viability of GS cells treated with different concentrations of AVP141-A by CCK-8 experiment.

[0022] Figure 2 It is a result diagram of the degree of virus infection and replication in cells after treatment with different AVP141-A concentrations.

[0023] Figure 3 It is a result diagram of detecting the transcriptional level (A) and protein level (B) of virus genes in cells after treatment with different AVP141-A concentrations.

[0024] Figure 4Results graph of virus infection and replication levels in cells treated with AVP141-A compared to the control group at 12 h and 24 h after SGIV infection.

[0025] Figure 5 Results graph of the transcriptional level (A), protein level (B), and titer (C) of viral genes in different cells treated with AVP141-A compared to the control group at 12 h and 24 h after SGIV infection. Detailed implementation method

[0026] 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 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 technical field.

[0027] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0028] Example 1

[0029] The extracellular polysaccharide AVP141-A of Aspergillus versicolor was prepared according to Patent CN115260336A.

[0030] Preparation of SGIV: Transfer grouper spleen (GS) cells to a 25 cm² culture flask. The cells grow in Leibovitz's L15 medium containing 10% fetal bovine serum (Gibco, Waltham, MA, USA) at a growth temperature of 28 °C. When the cells adhere and enter the logarithmic growth phase, add Singapore grouper iridovirus (SGIV) to the culture system according to a multiplicity of infection (MOI) of 0.5, and then place it in an incubator for culture. When most cells show obvious vacuolization, place the culture flask at -80 °C, freeze-thaw it 3 times, wait for the SGIV virus to be completely released, then aliquot the culture medium and store it at -80 °C.

[0031] To detect the toxic effect of AVP141-A at different concentrations on GS cells, a Cell Counting Kit-8 (CCK-8) was used to detect the effect of AVP141-A at different concentrations on the viability of cultured cells. GS cells were seeded in 96-well plates and cultured overnight at 28 °C. When the cells grew to confluence, the corresponding concentration of AVP141-A was added, and the plates were returned to the incubator at 28 °C with 5% CO2 for incubation. After 24 h of treatment, the cells were washed three times with fresh medium. 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 further cultured in an incubator at 28 °C for 1-4 h. The absorbance was measured at 450 nm using a multimode microplate reader (Thermo Fisher Science, USA).

[0032] It can be seen from Figure 1 that the cell viability of GS cells cultured in LeWBovitz's L15 medium containing different concentrations of AVP141-A was detected by the CCK-8 assay. The results showed that there was no significant change in the cell viability of GS cells treated with AVP141-A at concentrations of 1, 3, and 5 mg / mL for 24 h compared with the untreated group. The above results indicate that AVP141-A at concentrations of 1-5 mg / mL does not have a toxic effect on cells.

[0033] Example 2 Antiviral activity experiment of AVP141-A

[0034] GS cells were seeded in 24-well plates and cultured overnight at 28 °C. When the cells grew to confluence, AVP141-A at a non-toxic concentration to the cells was added. AVP141-A and GS cells were incubated in an incubator at 28 °C for 2 h, and then SGIV (MOI = 2) was added. After 24 h, the cytopathic effect of GS cells after adding different concentrations of AVP141-A was observed. Subsequently, total cellular RNA was extracted from the cells of different groups, and reverse transcription was performed 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:

[0035] 1. Extract cellular RNA using the Cell Total RNA Isolation Kit (Foregene, China). The specific steps are as follows:

[0036] (1) Add 250 μL of Buffer cRL1 to the collected cell samples.

[0037] (2) Transfer the lysed cell mixture to a DNA-Cleaning Column, then centrifuge at 12,000 rpm (=13,400×g) for 2 minutes. After that, remove the DNA-Cleaning Column and only retain the supernatant in the collection tube.

[0038] (3) Add Buffer cRL2 with a volume 1.6 times that of the supernatant obtained in step (2) to the supernatant, and then gently mix well.

[0039] (4) Transfer all the mixture to an RNA-only Column, centrifuge at 12,000 rpm (=13,400×g) for 1 minute, and discard the waste liquid in the collection tube.

[0040] (5) Add 500 μL of Buffer RW1 to the above purification column, then centrifuge at 12,000 rpm for 1 minute, and subsequently discard the waste liquid.

[0041] (6) Add 700 μL of Buffer RW2 to the above purification column, also centrifuge at 12,000 rpm for 1 minute, and then discard the waste liquid.

[0042] (7) Repeat the operation in step (6) again.

[0043] (8) Place the collection tube containing the purification column in an empty tube centrifuge at 12,000 rpm for 2 minutes.

[0044] (9) Transfer the purification column to a new EP tube, drop 30 - 50 μL of preheated RNase-Free ddH2O at 65°C onto the center of the purification column membrane, and then let it stand at room temperature for 2 minutes.

[0045] (10) Place the EP tube containing the above purification column in a centrifuge at 12,000 rpm for 2 minutes to collect the RNA solution.

[0046] (11) Take out 4 μL of the RNA sample for agarose gel electrophoresis detection, and take another 1 μL of the sample for concentration determination.

[0047] (12) Finally, place the remaining RNA in a water bath at 65°C for 5 minutes for pre-denaturation, and after the treatment, place it on ice.

[0048] (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.

[0049] 2. 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:

[0050] (1) Take 14 μL of the 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.

[0051] (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.

[0052] (3) Put the prepared reaction system into 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.

[0053] Table 1 Reverse transcription system

[0054]

[0055] Table 2 Reverse transcription program

[0056]

[0057] Next, according to the fluorescence quantitative system given in Table 3 and the fluorescence quantitative program shown in Table 4, with the help of fluorescence quantitative PCR and protein immunoblotting (Western blot) experiments, detect the transcription levels and protein levels of viral genes (MCP gene, ICP 18 gene, VP 19 gene) in different groups of cells respectively. The specific method steps are as follows:

[0058] 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 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.

[0059] Table 3 Fluorescent quantitative system

[0060]

[0061] Table 4 Fluorescent quantitative procedure

[0062]

[0063] 2. The specific operating steps of the protein immunoblotting (Western blot) experiment are as follows:

[0064] (1) Sample preparation: Collect the 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, boil in boiling water for 5 minutes to denature the protein, centrifuge at 12,000×g for 3 minutes, take the supernatant for electrophoresis, or store at -20°C for later use;

[0065] (2) Gel preparation: Pour the lower separating gel between clean glass plates. Its composition includes 30% acrylamide-bisacrylamide mixture (5.0 mL), 1.5 M Tris-HCl (pH 8.8) (2.6 mL), 10% SDS (0.1 mL), 10% ammonium persulfate (0.1 mL), and TEMED (0.01 mL). Make up to 10 mL with distilled water. After quickly pouring, carefully add a layer of isopropanol on the gel surface to prevent the gel surface from contacting with air and oxidizing. After the separating gel solidifies (about 30 - 60 minutes), pour out the upper liquid, dry the residual liquid with filter paper, and then pour the upper stacking gel. The formula for the stacking gel is 30% acrylamide-bisacrylamide mixture (0.6 mL), 1 M Tris-HCl (pH 6.8) (0.5 mL), 10% SDS (0.04 mL), 10% ammonium persulfate (0.04 mL), and TEMED (0.01 mL). Make up to 4 mL with distilled water. Insert the comb and wait for the stacking gel to solidify (about 30 - 40 minutes).

[0066] (3) Loading: Carefully pull out 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 measured into the sample wells. At the same time, add a protein Marker of appropriate size into the adjacent well to determine the molecular weight of the target protein.

[0067] (4) Electrophoresis: Electrophorese the upper gel at a low voltage of 70 V for 30 minutes, and the lower gel at a high voltage of 110 V for 60 minutes until the bromophenol blue indicator reaches the bottom of the gel.

[0068] (5) Transfer membrane: Soak the PVDF membrane (Millipore) in methanol for 1 - 2 minutes for activation. After the electrophoresis is completed, take out the protein gel, assemble the transfer membrane sandwich structure in the order of "sponge - filter paper - gel - membrane - filter paper - sponge", and gently roll out the air bubbles with a glass rod or roller. Place the transfer membrane sandwich into the transfer membrane device, and use the wet transfer method with a constant current of 100 mA for 60 minutes to transfer the protein blot in the protein gel to the PVDF membrane.

[0069] (6) Blocking: Put the transferred membrane into 5% skim milk prepared with PBST for blocking, and incubate it with slow shaking at room temperature for 2 - 3 hours, or overnight at 4°C.

[0070] (7) Primary antibody incubation: Take out the blocked membrane from the blocking solution and put it into the primary antibody solution diluted with the blocking solution. Incubate it with slow shaking at room temperature for 2 - 3 hours, or overnight at 4°C.

[0071] (8) Secondary antibody incubation: After the primary antibody incubation is completed, put the membrane into the PBST buffer solution, wash it with slow shaking at room temperature for 10 minutes each time, and wash it 3 times in total. Put the washed membrane into the secondary antibody solution diluted with the blocking solution. Incubate it with slow shaking at room temperature for 1 hour.

[0072] (9) Color development: Wash the membrane 3 times with the PBST buffer solution for 10 minutes each time to remove the unbound secondary antibody. Use the enhanced HRP - DAB substrate color development kit (TIANGEN) and perform color development according to the operation instructions.

[0073] (10) Photographing and analysis: Take pictures using a chemiluminescence imaging system, and perform gray - scale analysis on the pictures through Image J software.

[0074] After treating cells with different concentrations of AVP141 - A, the relevant results of the degree of virus infection and replication in cells can be referred to Figure 2 while after treating cells with different concentrations of AVP141 - A, the detection results of the transcriptional level and protein level of viral genes in cells can be viewed in Figure 3 .

[0075] It can be seen from Figure 2 that after treating cells with different concentrations (1, 3, 5 mg / mL) of AVP141 - A and infecting cells with SGIV, after treatment with AVP141 - A, the degree of cytopathic effect induced by SGIV is significantly reduced. The above results indicate that AVP141 - A has the effect of anti - SGIV.

[0076] It can be seen from Figure 3It can be seen that after treating cells with AVP141-A at different concentrations (1, 3, 5 mg / mL) and infecting the cells with SGIV, it was found that under the treatment conditions of AVP141-A at concentrations of 1, 3, and 5 mg / mL, both the transcriptional level and protein level of the SGIV virus MCP gene were significantly decreased. At the same time, the transcriptional levels of the ICP 18 gene and VP 19 gene in the SGIV virus were also significantly decreased; moreover, the inhibitory effect of AVP141-A on the transcription and protein expression of viral genes in cells was concentration-dependent. The above results indicate that AVP141-A has significant anti-SGIV activity.

[0077] Example 3

[0078] GS cells were treated with AVP141-A at a concentration of 5 mg / mL, and the effects of AVP141-A on the anti-SGIV activity of the cells were studied after different infection times with SGIV. Specifically, GS cells were inoculated in 12-well plates and cultured overnight at 28 °C. When the cells grew to confluence, AVP141-A at a concentration of 5 mg / mL was added. AVP141-A and GS cells were incubated in an incubator at 28 °C for 2 hours, and then SGIV (MOI = 2) was added. The cytopathic effects of GS cells co-incubated with AVP141-A were observed at 12 h and 24 h compared with the control group. Protein samples or RNA samples were collected to detect the transcriptional level of the MCP gene and the expression level of the protein.

[0079] At the same time, the cell samples and supernatants obtained from the above experimental groups were collected in the same centrifuge tube, frozen and thawed three times at -80 °C, and the virus titer in the cells was detected. The specific operation steps are as follows:

[0080] (1) Passage healthy GS cells into 96-well cell culture plates and culture overnight;

[0081] (2) After the cells evenly covered 80-90% of the bottom of the cell plate, the frozen and thawed virus-infected samples were taken out, melted and mixed well by shaking, and diluted 10-fold with serum-free medium. 100 μL of the gradient-diluted sample was added to each well of the cell plate;

[0082] (3) The virus diluted 10 1 ~10 11 was added to the 96-well plate, 100 μL per well, and an additional normal non-virus-infected control group was set up, with 8 parallels in each group;

[0083] (4) Observe the CPE caused by the virus every day, and record and calculate the TCID 50 of the sample according to the Reed-Muench method.

[0084] The results are asFigure 4 , Figure 5 As shown in Figure 5 , the inhibitory effects of AVP141-A on the transcription of the MCP gene and VP19 gene of SGIV virus were enhanced with the prolongation of SGIV infection time, and the effect of reducing the SGIV virus titer was also enhanced with the prolongation of infection time, indicating that the activity of AVP141-A in inhibiting SGIV infection was gradually enhanced with the prolongation of SGIV infection time.

Claims

1. Use of Aspergillus versicolor extracellular polysaccharide AVP141-A in the preparation of a drug against Singapore grouper iridovirus; the effective dose of Aspergillus versicolor extracellular polysaccharide AVP141-A in the drug is 1-5 mg / mL.

2. Use of Aspergillus versicolor extracellular polysaccharide AVP141-A in the preparation of a drug for preventing and / or treating fish diseases caused by Singapore grouper iridovirus infection; the effective dose of Aspergillus versicolor extracellular polysaccharide AVP141-A in the drug is 1-5 mg / mL.

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

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

5. The application according to claim 1 or 2, characterized in that, The effective dose of Aspergillus versicolor extracellular polysaccharide AVP141-A in the drug is 5 mg / mL.

6. The application according to claim 1 or 2, characterized in that The drug further contains a pharmaceutically acceptable excipient.

7. The application according to claim 6, wherein The excipient is selected from at least one of a pharmaceutically acceptable carrier, excipient or solvent.

8. The application 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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