Use of Saikosaponin D in the preparation of a drug against Pseudorabies virus in pigs

Abstract

The present invention discloses the use of saikosaponin D in the preparation of anti-pseudorabies virus drugs, belonging to the field of antiviral drug development. The experimental results show that saikosaponin D has strong anti-pseudorabies virus activity and can significantly inhibit the proliferation of pseudorabies virus at a concentration of 2 μM; saikosaponin D has pharmacological effects such as antipyretic, sedative, anti-inflammatory, antibacterial, liver-protecting, anti-nephritis, and immune regulation, with high safety, no obvious toxic and side effects, low drug residues and no pollution. Therefore, saikosaponin D has a broad application prospect in the preparation of anti-pseudorabies virus drugs.

Description

Technical Field

[0001] The present invention belongs to the field of antiviral applications, and particularly relates to the use of saikosaponin D in the preparation of drugs against porcine pseudorabies virus. Background Art

[0002] Pseudorabies (PR) is caused by Pseudorabies virus (PRV). Clinically, it mainly presents symptoms such as fever, itching, encephalomyelitis, and respiratory and nervous system disorders. Among them, the neurological symptoms of piglets are more obvious, and the mortality rate can reach 100%. Pigs are the natural hosts of PRV, but PRV can also infect a variety of domestic and wild animals, such as cattle, sheep, cats, rabbits, mice, dogs, minks, foxes, etc., causing fever, itching (except pigs) and encephalomyelitis. Currently, there are also reports of PRV infecting humans, indicating that PRV poses a potential threat to public health.

[0003] Pseudorabies virus is also known as Aujeszky's disease virus (ADV), belonging to the Herpesviridae family and the Alphaherpesvirinae subfamily. PRV is a double-stranded linear DNA virus with a genome length of about 150 kb and a GC content of over 70%. The PRV virion, like all other herpesvirus virions, has an icosahedral structure, consisting of core DNA, nucleocapsid, tegument, and envelope from the inside out. At the same time, there are many 8-10 nm spike proteins arranged radially on the envelope.

[0004] The transmission modes of PRV are horizontal and vertical. The virus can be transmitted through the respiratory tract, digestive tract, mating, semen, and placenta. PRV can infect pigs of all ages and causes great harm. It often makes the whole litter of piglets sick, showing neurological symptoms such as high fever, listlessness, dyspnea, tremors, and salivation. After fattening pigs are infected with PRV, they show symptoms such as slow growth, coughing, and fever. However, when PRV invades adult pigs, it generally has a latent course with a low mortality rate. When PRV invades sows, it will lead to breeding disorders. When pregnant sows are infected with PRV, they will have abortions, stillbirths, or mummified fetuses. PR can cause the testicles of boars to swell, atrophy, the semen quality to decline, the sexual function to decrease, and even the reproductive ability to be lost.

[0005] Saikosaponin D (SSD) is a saponin monomer component extracted from the dried roots of Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. of the Umbelliferae family. It is a kind of Bupleurum extract and one of the main active ingredients of Bupleurum. The research results on its therapeutic effect on non-alcoholic fatty liver disease (NAFLD) show that SSD dose-dependently alleviates high-fat diet-induced weight gain in mice, improves insulin sensitivity, and reduces liver lipid accumulation and injury-related biomarkers aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Current studies have shown that saikosaponin D has pharmacological effects such as antipyretic, sedative, anti-inflammatory, antibacterial, liver-protecting, anti-nephritis, and immune-regulating effects. However, there is no research report on saikosaponin D inhibiting pseudorabies virus yet.

[0006] The prevention and control of PRV mainly rely on inactivated vaccines and attenuated live vaccines. Antiviral drugs have been proven to play an important adjuvant therapeutic role in the infection process of various viral diseases. The first case of pseudorabies virus was reported in China in the 1950s, and the Bartha-K61 vaccine was introduced into China in the 1970s, which has played an important role in the prevention and control of PRV in China. However, with the emergence of PRV variant strains, the immune protection effect of the original vaccines has decreased to a certain extent. Therefore, the development of effective PRV therapeutic drugs has great prospects for the prevention, control, and adjuvant treatment of PRV.

[0007]

[0008] Saikosaponin D Summary of the Invention

[0009] In the prior art, there is no research report on saikosaponin D inhibiting pseudorabies virus. The purpose of the present invention is to provide a pharmaceutical composition against pseudorabies virus, and to provide the use of saikosaponin D in the preparation of drugs against pseudorabies virus.

[0010] The purpose of the present invention can be achieved by the following technical solutions:

[0011] In the first aspect of the present invention, there is provided the use of saikosaponin D in the preparation of drugs against porcine pseudorabies virus.

[0012] Furthermore, the saikosaponin D is used to inhibit the infection of animal pseudorabies virus.

[0013] Furthermore, the saikosaponin D is used to inhibit the proliferation of pseudorabies virus after infection.

[0014] Furthermore, the concentration of the saikosaponin D is 2-10 μM.

[0015] Furthermore, the saikosaponin D is used to inhibit the adsorption stage of pseudorabies virus.

[0016] Furthermore, fluorescence quantitative PCR was used to measure the change in the genomic content of pseudorabies virus during the adsorption stage, and the primer sequences were as follows:

[0017] Pig GAPDH-F: 5'-CCTTCATTGACCTCCACTACA-3';

[0018] Pig GAPDH-R: 5'-GATGGCCTTTCCATTGATGAC-3';

[0019] PRV-UL42-F: 5'-GACCGTCTTCAACGTCACCT-3';

[0020] PRV-UL42-R: 5'-GCATGATGCAGTAGTCGTTG-3'.

[0021] Furthermore, the saikosaponin D is used to inhibit the entry stage of pseudorabies virus.

[0022] Furthermore, fluorescence quantitative PCR was used to measure the change in the genomic content of pseudorabies virus during the entry stage, and the primer sequences were as follows:

[0023] PRV-UL42-F: 5'-CACGTGGGTCAAGCTCATC-3';

[0024] PRV-UL42-R: 5'-CATCACCAGGGTGTAGGTGA-3'.

[0025] In the second aspect of the present invention, an anti-pseudorabies virus drug for pigs includes saikosaponin D and also includes pharmaceutically acceptable excipients or auxiliary components.

[0026] In the above anti-pseudorabies virus drug for pigs, the mass percentage content of the saikosaponin D is not less than 99.50%, and its application dose is 2 - 10 μM.

[0027] In some preferred embodiments, the anti-pseudorabies virus drug for pigs can be tablets, powders, granules, capsules, oral liquids, injections or sustained-release agents.

[0028] Optionally, the pseudorabies virus for pigs is a genetically variant pseudorabies virus.

[0029] Optionally, to prepare the pharmaceutical composition of the present invention, saikosaponin D can be mixed with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers that can be used in this composition cover any standard pharmaceutical carriers, such as phosphate buffer aqueous solutions, carbonate buffers, water and emulsions and other preparations.

[0030] Beneficial effects

[0031] Saikosaponin D has a very significant effect against Pseudorabies virus in pigs; experiments have shown that saikosaponin D exhibits obvious antiviral activity in cell experiments and can show a strong inhibitory effect against Pseudorabies virus in pigs (MOI = 0.15) at a concentration of 2 μM.

[0032] Saikosaponin D is safe for use against Pseudorabies virus in pigs and has few toxic and side effects; saikosaponin D is an extract from Chinese herbal medicines, different from hormones, antibiotics, chemically synthesized drugs, etc., and has no obvious toxic and side effects on the body.

[0033] Saikosaponin D has low drug residues and no pollution when used against Pseudorabies virus in pigs; saikosaponin D belongs to an organic molecular compound, is easily absorbed by the animal body, has a high biological metabolism rate, and has no pollution during excretion. Description of the Drawings

[0034] Figure 1 Results of detecting the toxicity of saikosaponin D to PK15 cells by the CCK-8 method. PK15 cells were treated with 2.5, 4, 5, 7.5, 8, 10, 12, 16, 18, 20, 25, 30, 35, 40, 45, 50 μM saikosaponin D for 24 hours, then CCK-8 solution was added for color development for 3 hours, and the cell absorbance was measured at 450 nm to detect cell viability.

[0035] Figure 2 Results of determining the inhibitory effect of saikosaponin D on Pseudorabies virus on PK15 cells by the Cell-based ELISA method. PK15 cells were pretreated with 0.2, 0.5, 0.8, 1.0, 1.6, 2, 2.4, 2.8, 3.2, 3.6 μM saikosaponin D for 2 hours, then infected with PRV (0.15 MOI) at the same drug concentration, the cells were fixed after 24 hours, incubated with anti-PRV-UL42 protein primary antibody and HRP-labeled rabbit anti-mouse secondary antibody, and the absorbance was measured at 450 nm after color development to calculate the Pseudorabies virus infection rate. The data was analyzed to obtain the concentration of saikosaponin D required to inhibit 50% of the virus (IC 50 ).

[0036] Figure 3 Results of determining the inhibitory effect of saikosaponin D on Pseudorabies virus infection on PK15 cells by Western blot. PK15 cells were pretreated with 0.5 - 2 μM saikosaponin D for 2 hours, then infected with Pseudorabies virus (0.15 MOI), and saikosaponin D was present throughout. After 24 hours, the cells were harvested for Western blot to determine the content of PRV-UL42 virus protein in the cells.

[0037] Figure 4For the plaque formation assay to determine the inhibitory effect of saikosaponin D on the adsorption stage of pseudorabies virus in PK15 cells. PK15 cells were pretreated with 2 μM saikosaponin D for 2 hours, then inoculated with pseudorabies virus (5 MOI) and infected at 4°C for 1 hour. During this period, saikosaponin D was present all the time. The cell pellet was collected and the virus titer was determined by plaque formation assay.

[0038] Figure 5 For the fluorescence quantitative PCR to determine the inhibitory effect of saikosaponin D on the adsorption stage of pseudorabies virus infection in PK15 cells. PK15 cells were pretreated with 2 μM saikosaponin D for 2 hours, then infected with pseudorabies virus (5 MOI) and incubated at 4°C for 1 hour. During this period, saikosaponin D was present all the time. Finally, the cells were collected to extract total RNA, and the expression level of PRV-UL42 gene was determined by fluorescence quantitative PCR.

[0039] Figure 6 For the plaque formation assay to determine the inhibitory effect of saikosaponin D on the entry stage of pseudorabies virus in PK15 cells. PK15 cells were pretreated with 5 μM saikosaponin D for 2 hours, infected with pseudorabies virus (5 MOI) and incubated at 4°C for 1 hour, and then the cells were incubated at 37°C for 1 hour. During this period, saikosaponin D was present all the time. The cell pellet was collected and the virus titer was determined by plaque formation assay.

[0040] Figure 7 For the fluorescence quantitative PCR to determine the inhibitory effect of saikosaponin D on the entry stage of pseudorabies virus infection in PK15 cells. PK15 cells were pretreated with 5 μM saikosaponin D for 2 hours, then infected with pseudorabies virus (5 MOI) and incubated at 4°C for 1 hour, and then the cells were incubated at 37°C for 1 hour. During this period, saikosaponin D was present all the time. Then the cells were collected to extract total RNA, and the expression level of PRV-UL54 gene was determined by fluorescence quantitative PCR.

[0041] Figure 8 For the indirect immunofluorescence to determine the inhibitory effect of saikosaponin D on pseudorabies virus infection in PK15 cells. PK15 cells were pretreated with saikosaponin D at concentrations of 0.5 - 2 μM for 2 hours and then infected with pseudorabies virus (0.15 MOI). After that, saikosaponin D was present all the time. After 18 hours, the cells were fixed, blocked with 3% BSA, incubated with the primary antibody against PRV-UL42 protein and the secondary antibody FITC-conjugated goat anti-mouse IgG; stained with DAPI (containing anti-fluorescence quencher) in the dark at room temperature. Finally, the cells were observed with the light-conducting coupled illumination fluorescence light source U-HGLGPS. Detailed implementation mode

[0042] Abbreviation

[0043] Saikosaponin D: SSD

[0044] Pseudorabies virus: PRV

[0045] Test materials

[0046] The JSY13 strain of porcine pseudorabies virus is owned by the inventor's laboratory;

[0047] PK-15 (porcine kidney cells) and Vero (African green monkey kidney cells) were purchased from ATCC and stored in the inventor's laboratory;

[0048] The CCK-8 detection kit was purchased from Apexbio, USA;

[0049] The Real time PCR AceQGreen Master Mix kit was purchased from Novoprotein;

[0050] Saikosaponin D (SSD) was purchased from Selleck, with a purity of 99.50%.

[0051] Example 1

[0052] Determination of the cytotoxicity of saikosaponin D on PK15 cells

[0053] PK-15 cells were seeded in 96-well plates (2×10 4 cells / well), placed in an incubator at 37°C and 5% CO2 overnight for cell attachment. The cell culture medium was discarded, and saikosaponin D was diluted with DMEM containing 10% FBS to final concentrations of 2.5, 4, 5, 7.5, 8, 10, 12, 16, 18, 20, 25, 30, 35, 40, 45, 50 μM. 100 μL / well was added to PK15 cells, and three replicates were set for each concentration. At the same time, a DMSO negative control group was set. Under the conditions of 37°C and 5% CO2, the cells were further cultured for 24 hours; then 10 μL of CCK-8 solution was added to each well, and the incubation was continued for 3 hours. The absorbance was measured at 450 nm. The data was analyzed according to the CCK-8 kit instructions to finally obtain the minimum cytotoxic concentration of the drug, that is, the minimum concentration required to kill 50% of the cells (CC 50 ), and the results are shown in the appendix Figure 1 as shown. As can be seen from the appendix Figure 1 , the CC 50 value of saikosaponin D on PK-15 cells was 14.430 μM. When the concentration of saikosaponin D was 2 μM, it had no obvious cytotoxicity to PK-15 cells (cell survival rate > 95%).

[0054] Example 2

[0055] Determination of the inhibitory effect of saikosaponin D on pseudorabies virus using the Cell-based ELISA method

[0056] Seed PK15 cells into a 96-well plate at a density of 2×10 4 / well, and culture overnight in a 37°C, 5% CO2 cell culture incubator. Discard the culture medium, dilute the drug to 0.2, 0.5, 0.8, 1.0, 1.6, 2, 2.4, 2.8, 3.2, 3.6 μM with 4% FBS DMEM, add 100 μL / well to the wells, set 3 wells for each concentration, and pretreat the cells for 2 hours at 37°C, 5% CO2; at the same time, set a negative control well without adding the drug; dilute PRV JSY13 to 0.15 MOI with serum-free DMEM, add 100 μL / well to the wells, and set a positive control well without virus infection at the same time. Infect at 37°C, 5% CO2 for 24 hours; saikosaponin D has been present in the corresponding wells at the same concentration; discard the culture medium, gently rinse once with PBS, add 100 μL / well of fixative (PBS containing 4% paraformaldehyde and 0.1% Triton X-100), fix at 37°C for 30 minutes, discard the fixative, rinse once with 200 μL / well of Glycine-PBS (PBS containing 20 mM Glycine), and block the cells with 3% skim milk at 37°C for 2 hours; dilute the primary antibody against PRV-UL42 protein, incubate at 100 μL / well at 37°C for 2 hours, and rinse 5 times with PBST; dilute the HRP-labeled rabbit anti-mouse secondary antibody, incubate at 100 μL / well at 37°C for 1 hour, and rinse 5 times with PBST; mix the TMB two-component chromogenic solution 1:1 and add 100 μL / well to develop color in the dark for 15 minutes, add 50 μL / well of 2M sulfuric acid termination solution to terminate the color development, and place the well plate on an enzyme-linked immunosorbent assay (ELISA) reader to read the absorbance value at 450 nm. Analyze the obtained OD 450 to finally obtain the concentration required to inhibit 50% of the virus (IC 50 ). The results are as shown in Figure 2 . The IC 50 of saikosaponin D is 1.692 μM, that is, 1.692 μM of saikosaponin D can inhibit 50% of the virus.

[0057] Example 3

[0058] Determination of the activity of saikosaponin D in inhibiting pseudorabies virus infection on PK15 cells by Western blot

[0059] Seed PK-15 cells at 6×10 5Cells were seeded at a density of [X] cells / well in a 6-well plate and cultured overnight at 37°C in a 5% CO₂ incubator. The culture medium was discarded, and saikosaponin D was diluted to 0.5 μM, 1 μM, 1.5 μM, and 2 μM with DMEM containing 4% FBS, and 1 mL / well was used to pretreat the cells for 2 hours. The PRV JSY13 strain was diluted to 0.15 MOI with DMEM and added to the cells pretreated with saikosaponin D at 1 mL / well. At the same time, a negative control (MOCK) without drug addition and without virus inoculation and a positive control without drug addition but with virus inoculation were set up. After 24 hours, the culture medium was discarded, and the cells were washed once with PBS. The cells were lysed with 2×SDS sample buffer and boiled at 96°C for 10 minutes. The treated samples were taken for SDS-PAGE and transferred to a BioTrace NT Nitrocellulose (NC) membrane. The membrane was blocked with 3% skim milk at room temperature for 2 hours, then incubated with PRV-UL42 antibody and Actin antibody overnight at 4°C. The membrane was washed 3 times with TBST. Then it was incubated with HRP-labeled rabbit anti-mouse secondary antibody for 4 hours at 4°C, and the membrane was washed 3 times with TBST. The signal was visualized using a high-sensitivity ECL chemiluminescence kit through a Tanon 5200 system. The results are shown in Figure 3 As shown, it shows that when the concentration reaches 2 μM, saikosaponin D can significantly inhibit the infection of porcine pseudorabies virus.

[0060] Example 4

[0061] Determination of the inhibition of saikosaponin D on the adsorption stage of porcine pseudorabies virus by plaque formation assay

[0062] PK15 cells were seeded at a density of 6×10 5 cells / well in a 6-well plate and cultured in a cell culture incubator until they adhered. Saikosaponin D was diluted to 2 μM (1 mL / well) with DMEM containing 10% FBS and treated at 37°C in a 5% CO₂ incubator for 2 hours. The culture medium was discarded, and then a virus inoculum containing JSY13 (5 MOI) and 2 μM saikosaponin D was prepared with serum-free DMEM and added to the cells at 1 mL / well. The cells were incubated at 4°C for 1 hour. At the same time, a control group without drug addition (MOCK) was set up. The culture medium was discarded, and the cells were gently washed 3 times with pre-cooled PBS. When washing for the last time, the PBS was discarded completely. 1 mL of DMEM was added, and the cells were repeatedly frozen and thawed, and then the cell pellet was collected. The harvested virus solution was serially diluted and inoculated into a 6-well plate seeded with a monolayer of Vero cells. The plate was incubated in a 37°C, 5% CO₂ incubator for 2 hours, washed 3 times with PBS, and then 2 mL of a 1:1 mixture of 2×DMEM containing 4% fetal bovine serum and 2% low melting point agarose was added. The plate was incubated in a 37°C, 5% CO₂ incubator for 2 - 3 days, and the formation of plaques was observed. When obvious visible plaques appeared, 1% crystal violet was added for staining. The results are shown in Figure 4As shown, it is shown that saikosaponin D can significantly inhibit the adsorption process of pseudorabies virus.

[0063] Example 5

[0064] Inhibition of saikosaponin D on the adsorption stage of PRV on PK-15 cells determined by fluorescence quantitative PCR (qRT-PCR)

[0065] Seed PK15 cells at 6x10 5 cells per well in a 6-well plate and culture them in a cell incubator until they adhere. Dilute saikosaponin D to 2 μM (1 mL per well) with DMEM containing 10% FBS and treat for 2 hours at 37°C under 5% CO2; discard the culture medium, then prepare an inoculum containing 5 MOI JSY13 and 2 μM saikosaponin D with serum-free DMEM, add 1 mL per well to the cells, and incubate at 4°C for 1 hour. At the same time, set up a non-drug control group (MOCK); discard the culture medium, then gently wash the cells 3 times with pre-cooled PBS, discard the PBS completely in the last wash, add 1 mL of Trizol, extract RNA, reverse transcribe it into cDNA, and use real-time fluorescence quantitative PCR to determine the change in the PRV genome content.

[0066] Among them, the primer information is as follows:

[0067] Pig GAPDH-F: 5'-CCTTCATTGACCTCCACTACA-3';

[0068] Pig GAPDH-R: 5'-GATGGCCTTTCCATTGATGAC-3';

[0069] PRV-UL42-F: 5'-GACCGTCTTCAACGTCACCT-3';

[0070] PRV-UL42-R: 5'-GCATGATGCAGTAGTCGTTG-3'.

[0071] The fluorescence quantitative PCR system is as follows:

[0072]

[0073] qRT-PCR is carried out on a LineGene 9600Plus fluorescence quantitative polymerase reaction (PCR) detection system, and the qRT-PCR reaction is carried out under the following conditions:

[0074] Pre-denaturation at 95°C for 5 min, PCR amplification at 95°C for 10 s, 60°C for 30 s, melting curve at 95°C for 15 s, 60°C for 60 s, 95°C for 15 s. Then perform 40 cycles at 95°C for 10 s and 60°C for 30 s.

[0075] This experiment was carried out by -ΔΔCt the 2 Figure 5 method for relative quantification of the PRV-UL42 gene. The results are shown in the appendix

[0076] Example 6

[0077] Plaque formation assay for determining the inhibition of saikosaponin D on the entry stage of pseudorabies virus

[0078] Seed PK15 cells at 6x10 5 cells / well in a 6-well plate, wash 3 times with PBS, then dilute PRV to 5 MOI with serum-free DMEM, 1 mL / well, and incubate at 4°C for 1 hour; wash the cells 3 times with PBS, dilute saikosaponin D to 5 mM with 2% FBS DMEM, add 1 mL / well to the cells, set up a non-drug control group (MOCK), and incubate in a 37°C incubator for 1 hour; wash the cells 3 times with sodium citrate at pH = 3, then wash 3 times with PBS, and then add 1 mL DMEM to collect cell samples for virus titer determination by virus plaque assay. The results are shown in the appendix Figure 6 as shown, indicating that saikosaponin D can significantly inhibit the entry stage of porcine pseudorabies virus.

[0079] Example 7

[0080] Fluorescent quantitative PCR (qRT-PCR) for determining the inhibition of saikosaponin D on the entry stage of pseudorabies virus in PK-15 cells

[0081] Seed PK15 cells at 6x10 5 cells / well in a 6-well plate and incubate overnight adherently, wash 3 times with PBS, then dilute PRV to 5 MOI with serum-free DMEM, 1 mL / well, and incubate at 4°C for 1 hour; wash the cells 3 times with PBS, dilute saikosaponin D to 5 mM with 2% FBS DMEM, add 1 mL / well to the cells, set up a non-drug control group (MOCK), and incubate in a 37°C incubator for 1 hour; wash the cells 3 times with sodium citrate at pH = 3, then wash 3 times with PBS, and then add 1 mL Trizol to extract RNA, reverse transcribe it into cDNA, and use qRT-PCR to determine the changes in Pig GAPDH and PRV UL54 mRNA.

[0082] Among them, the primer information is as follows:

[0083] PRV-UL42-F: 5'-CACGTGGGTCAAGCTCATC-3';

[0084] PRV-UL42-R: 5'-CATCACCAGGGTGTAGGTGA-3'.

[0085] The results are shown in the Figure 7 attachment, indicating that saikosaponin D can significantly inhibit the entry stage of pseudorabies virus.

[0086] Example 8

[0087] Indirect immunofluorescence assay for the inhibition of pseudorabies virus infection by saikosaponin D on PK15 cells

[0088] Place sterile cover slips in six-well plates. Seed PK15 cells at 6x10 5 cells / well in six-well plates and culture overnight at 37°C and 5% CO2. Dilute saikosaponin D to 0.5 μM, 1 μM, 1.5 μM, and 2 μM respectively with DMEM containing 4% FBS, and pretreat the cells with 1 mL / well for 2 hours. Dilute JSY13 to 0.15 MOI with DMEM and add 1 mL / well to the cells pretreated with saikosaponin D. At the same time, set up a negative control (MOCK) without adding drugs or virus and a positive control (POSITIVE) without adding drugs but only virus. After 18 hours, discard the culture medium, gently wash the cells once with PBS, and fix the cells with fixative (PBS containing 4% paraformaldehyde and 0.1% Triton X-100) at 1 mL / well for 30 minutes at 37°C. Rinse twice with Glycine-PBS (PBS containing 20 mM Glycine), and then rinse 3 times with PBS; block with 3% BSA at 1 mL / well for 30 minutes at 37°C, rinse once with PBS, and suck out the liquid around the cover slips. Incubate with PRV-UL42 protein antibody, 100 μL / well, and incubate the cells at 37°C for 1 hour, then wash the cells three times with PBS. Incubate with FITC-labeled goat anti-mouse IgG secondary antibody, 100 μL / well, and incubate at 37°C for 30 minutes, then wash the cells 3 times with PBS. Stain the cell nuclei with DAPI (containing anti-fluorescence quencher) in the dark at room temperature. Finally, observe the cells with a photoconductive coupled illumination fluorescence light source U-HGLGPS. The results are shown in the Figure 8 attachment, showing that saikosaponin D can significantly inhibit the proliferation of pseudorabies virus. When the concentration is 1 μM, a certain degree of inhibition appears, and then the inhibitory effect gradually becomes obvious with the increase of concentration.

[0089] The above is the preferred embodiment of the present invention, but the present invention should not be limited to the content disclosed in this embodiment. Therefore, all equivalent or modified implementations completed without departing from the spirit disclosed by the present invention fall within the protection scope of the present invention.

Claims

1. Use of saikosaponin D in the preparation of a drug against porcine pseudorabies virus.

2. The use according to claim 1, characterized in that, The saikosaponin D is used to inhibit the infection of animal pseudorabies virus.

3. The use according to claim 1, wherein The saikosaponin D is used to inhibit the proliferation of pseudorabies virus after infection.

4. The use according to claim 1, characterized in that, The concentration of the saikosaponin D is 2 - 10 μM.

5. The use according to claim 1, characterized in that, The saikosaponin D is used to inhibit the adsorption stage of pseudorabies virus.

6. The use according to claim 5, characterized in that, Fluorescence quantitative PCR is used to measure the change in the genomic content of pseudorabies virus at the adsorption stage, and the primer sequences are as follows: Pig GAPDH-F: 5'-CCTTCATTGACCTCCACTACA-3'; Pig GAPDH-R: 5'-GATGGCCTTTCCATTGATGAC-3'; PRV-UL42-F: 5'-GACCGTCTTCAACGTCACCT-3'; PRV-UL42-R: 5'-GCATGATGCAGTAGTCGTTG-3'.

7. The use according to claim 1, characterized in that, The saikosaponin D is used to inhibit the entry stage of pseudorabies virus.

8. The use according to claim 7, characterized in that, Fluorescence quantitative PCR is used to measure the change in the genomic content of pseudorabies virus at the entry stage, and the primer sequences are as follows: PRV-UL42-F: 5'-CACGTGGGTCAAGCTCATC-3'; PRV-UL42-R: 5'-CATCACCAGGGTGTAGGTGA-3'.

Images