An anti-swine pseudorabies virus infection preparation and application of radix rehmanniae polysaccharides in preparation of the preparation

Abstract

This invention relates to an anti-swine pseudorabies virus (PRV) preparation and the application of Rehmannia glutinosa polysaccharide in its preparation, belonging to the field of biopharmaceutical technology. This invention provides an anti-swine pseudorabies virus (PRV) preparation comprising Rehmannia glutinosa polysaccharide. It also provides the application of Rehmannia glutinosa polysaccharide in the preparation of an anti-PRV preparation for the prevention and / or treatment of swine pseudorabies. This invention utilizes Rehmannia glutinosa polysaccharide to prevent infection by the PRV variant XJ5 by reducing intracellular oxidative stress levels; it also resists viral infection by affecting viral adsorption. Furthermore, when the concentration of Rehmannia glutinosa polysaccharide is between 50 and 400 μg / mL, it can also affect viral entry and replication, and has no cytotoxicity to PK-15 cells.

Description

Technical Field

[0001] This invention belongs to the field of biological agent technology, and in particular relates to an anti-swine pseudorabies virus infection agent and the application of Rehmannia glutinosa polysaccharide in the preparation of the agent. Background Technology

[0002] Pseudorabies (PR) is an acute infectious disease of pigs caused by the pseudorabies virus (PRV), initially transmitted from cattle to pigs. With the expansion of intensive pig farms, the globalization of international trade in animals and their byproducts, and the increased demand for domestic pig transportation, the disease has experienced explosive outbreaks. The disease is transmitted through the mouth and nose, subsequently invading the respiratory tract and tonsils. Affected pigs exhibit high fever, chills, depression, hoarseness, and significantly reduced food and water intake. Damage to the central nervous system leads to cerebral sulci hemorrhage, resulting in ataxia during gait, pulmonary edema, enlarged tonsils, and white necrotic spots on the surface of the liver, kidneys, and spleen. Major symptoms include abortion and stillbirth in pregnant sows, mummified fetuses, repeated infertility and high rates of return to estrus, infertility in boars, and high neurological symptoms and mortality rates in newborn piglets. In finishing pigs, in addition to typical clinical symptoms, infection primarily affects weight gain, causing stunted growth and significant losses to farms. my country imported the Bartha-K61 vaccine from Hungary in 1987 to control porcine pseudorabies virus. In the following years, domestically produced Bartha-K61 vaccines also appeared, and with their application, porcine pseudorabies was effectively controlled. However, with the emergence of multiple variant strains in 2011, the widely used Bartha-K61 vaccine only provided 50%–60% protection to pigs, and the mortality rate of 12-week-old pigs approached 100%. The variant strains showed abnormally increased virulence in pigs. There is no effective treatment for this disease; prevention is the primary focus in the early stages, achieved through the purchase of imported Bartha-K61 vaccine (with a median lethal dose of 10). 5 To achieve the goal of eliminating wild-type strains and ultimately purifying the pseudorabies-infected pig population, a "carpet-style" immunization of pig herds is necessary. However, since there is currently no specific drug for the prevention and treatment of this disease, and the prevalence of pseudorabies in my country is on the rise, any outbreak of pseudorabies in pig herds will bring direct and indirect economic losses to the development of my country's pig farming industry.

[0003] PRV belongs to the alpha-herpesvirus family. Currently, almost half of the PRV gene product is the structure of a mature virion, which gives it strong pathogenicity. Meanwhile, porcine pseudorabies virus is a highly latent infection virus, easily causing outbreaks due to changes in the internal and external environment and stress factors. Virulence is controlled by multiple genes working together. PRV has a wide host range, a short replication cycle, and high pathogenicity. Structurally, it consists of four layers from the inside out: double-stranded DNA, nucleocapsid, protein, and envelope. The UL region of the PRV genome contains multiple virulence genes, such as gB and gC, which, along with other genes, determine the virus's virulence. The gB gene, 2742 bp in length, is the main virulence gene of PRV, inducing the body to produce two types of neutralizing antibodies, enabling PRV particles to fuse with host cells and complete the invasion process. Most vaccines currently on the market are those lacking virulence genes. Currently, there are several vaccines developed in the domestic market using classic strains as parents. Antibody detection kits can effectively distinguish between immune antibodies and wild-type virus infection antibodies, but due to novel mutations at the viral genome level... Traditional vaccines cannot completely prevent swine pseudorabies (PR), making the development of a biological agent for the prevention and / or treatment of the currently prevalent variant of swine pseudorabies urgent and necessary. This agent avoids the first-pass effect in the liver and gastrointestinal inactivation that can occur with oral administration, thus avoiding many side effects.

[0004] Rehmannia glutinosa polysaccharide is a product made from the processed and steamed root of Rehmannia glutinosa. As a traditional Chinese medicine, it has been reported since the last century to have positive effects in inhibiting tumor growth, promoting lymphocyte proliferation, and stimulating hematopoietic stem cells. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to design and provide an anti-swine pseudorabies virus infection preparation and the application of Rehmannia glutinosa polysaccharide in the preparation of the preparation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An anti-swine pseudorabies virus infection preparation, wherein the anti-swine pseudorabies virus infection preparation comprises Rehmannia glutinosa polysaccharide.

[0008] The aforementioned preparation for combating swine pseudorabies virus infection includes, among other types, drugs, feed additives, and disinfectants; the concentration of Rehmannia glutinosa polysaccharide in the preparation is 50–400 μg / mL, preferably 400 μg / mL.

[0009] Application of Rehmannia glutinosa polysaccharide in the preparation of anti-pseudorabies virus agents for the prevention and / or treatment of porcine pseudorabies.

[0010] In the aforementioned application, the concentration of Rehmannia glutinosa polysaccharide in the anti-swine pseudorabies virus infection preparation is 50–400 μg / mL, preferably 400 μg / mL.

[0011] The application described herein is that the anti-pseudorabies virus infection agent prevents and / or treats swine pseudorabies by inhibiting the adsorption of swine pseudorabies virus.

[0012] In the aforementioned application, the anti-swine pseudorabies virus infection preparation achieves its effect of preventing infection by reducing intracellular oxidative stress levels.

[0013] The application described herein is that the anti-pseudorabies virus infection agent prevents and / or treats pseudorabies by inhibiting the entry of the pseudorabies virus into cells.

[0014] The application described herein is that the anti-pseudorabies virus infection agent prevents and / or treats swine pseudorabies by inhibiting the replication of swine pseudorabies virus.

[0015] Application of Rehmannia glutinosa polysaccharide in the preparation of antioxidant agents.

[0016] In the aforementioned application, the prepared Rehmannia polysaccharide reduces oxidative stress by inhibiting the production of reactive oxygen species.

[0017] Compared with other drugs with antiviral effects, the anti-swine pseudorabies virus preparation of the present invention has the following advantages:

[0018] 1. The Rehmannia glutinosa polysaccharide component in the anti-pseudorabies virus preparation of this invention has low environmental pollution after being metabolized by the body.

[0019] 2. Rehmannia glutinosa polysaccharide has a significant effect on resisting the infection of cells by the XJ5 strain of porcine pseudorabies virus. Experiments have shown that this drug can reduce the cytopathic effects caused by the virus.

[0020] 3. When the concentration of Rehmannia glutinosa polysaccharide is between 50 and 400 μg / mL, it can prevent infection by the variant strain XJ5 of porcine pseudorabies virus by reducing intracellular oxidative stress; it also resists viral infection by affecting viral adsorption. Simultaneously, the concentration of Rehmannia glutinosa polysaccharide at this concentration can also affect viral entry and replication, and has no cytotoxicity to PK-15 cells. Attached Figure Description

[0021] Figure 1 To observe under a microscope the effect of Rehmannia glutinosa polysaccharide on cytopathic effects induced by the porcine pseudorabies virus variant XJ5 on PK-15 cells;

[0022] Figure 2The results show the effect of Rehmannia glutinosa polysaccharide on the infection of porcine pseudorabies virus variant XJ5 in PK-15 cells; where A is the effect of Rehmannia glutinosa polysaccharide on the infection of porcine pseudorabies virus variant XJ5 in PK-15 cells determined by Western blot; B is the grayscale scan analysis of gB protein in Figure A; C is TCID. 50 The effect of Rehmannia glutinosa polysaccharide on infection of porcine pseudorabies virus variant XJ5 in PK-15 cells was determined; D represents the effect of Rehmannia glutinosa polysaccharide on infection of porcine pseudorabies virus variant XJ5 in PK-15 cells by immunofluorescence antibody assay (IFA).

[0023] Figure 3 The results show the effect of Rehmannia glutinosa polysaccharide on the adsorption and cell entry of porcine pseudorabies virus variant XJ5 in PK-15 cells; where A is the effect of Western blot on the adsorption and cell entry of Rehmannia glutinosa polysaccharide on porcine pseudorabies virus variant XJ5 in PK-15 cells; B is the grayscale scan result of gB protein in Figure A; C is TCID. 50 The effect of Rehmannia glutinosa polysaccharide on adsorption and cell entry of porcine pseudorabies virus variant XJ5 on PK-15 cells was determined; D represents the effect of immunofluorescence antibody assay (IFA) on adsorption and cell entry of Rehmannia glutinosa polysaccharide on porcine pseudorabies virus variant XJ5 on PK-15 cells.

[0024] Figure 4 The results show the effect of Rehmannia glutinosa polysaccharide on the adsorption of porcine pseudorabies virus variant XJ5 on PK-15 cells; where A is the effect of Western blot determination on the adsorption of Rehmannia glutinosa polysaccharide on porcine pseudorabies virus variant XJ5 on PK-15 cells; B is the grayscale scan analysis of gB protein in Figure A; C is TCID. 50 The effect of Rehmannia glutinosa polysaccharide on the adsorption of porcine pseudorabies virus variant XJ5 on PK-15 cells was determined; D represents the effect of immunofluorescence assay (IFA) on the adsorption of Rehmannia glutinosa polysaccharide on porcine pseudorabies virus variant XJ5 on PK-15 cells.

[0025] Figure 5 The results show the effect of Rehmannia glutinosa polysaccharide on the cell entry of porcine pseudorabies virus variant XJ5 in PK-15 cells; where A represents the effect of Western blot on the cell entry of Rehmannia glutinosa polysaccharide on porcine pseudorabies virus in PK-15 cells; B represents... Figure 5 A represents the result obtained from grayscale analysis; C represents TCID. 50 The effect of Rehmannia glutinosa polysaccharide on the entry of porcine pseudorabies virus variant XJ5 into PK-15 cells was determined; D represents the effect of Rehmannia glutinosa polysaccharide on the entry of porcine pseudorabies virus variant XJ5 into PK-15 cells by immunofluorescence assay (IFA).

[0026] Figure 6 The results of Western blot analysis of the effect of Rehmannia glutinosa polysaccharide on the replication of the porcine pseudorabies virus variant XJ5 in PK-15 cells are shown. Figure A shows the effect of Western blot analysis of Rehmannia glutinosa polysaccharide on the replication of porcine pseudorabies virus in PK-15 cells 4 hours after infection; Figure B shows the grayscale analysis of the Western blot results; Figure C shows the effect of Western blot analysis of Rehmannia glutinosa polysaccharide on the replication of porcine pseudorabies virus in PK-15 cells 6 hours after infection; and Figure D shows the grayscale analysis results of Figure C.

[0027] Figure 7 To detect the effect of Rehmannia glutinosa polysaccharide on reducing oxidative stress in PK-15 cells by flow cytometry analysis;

[0028] Figure 8 The results of cytotoxicity assays of Rehmannia glutinosa polysaccharide on PK-15 cells were obtained using the CCK8-Kit. Detailed Implementation

[0029] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0030] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0031] Example 1: Formulation of an anti-swine pseudorabies virus preparation

[0032] 0.05g of Rehmannia glutinosa polysaccharide powder purchased from Yangling Ciyuan Biotechnology Co., Ltd. was weighed and dissolved in 1mL of PBS solution to obtain an anti-swine pseudorabies virus preparation.

[0033] Example 2: Rehmannia glutinosa polysaccharide inhibits infection of mutant porcine pseudorabies virus XJ5.

[0034] PK-15 cells revived from the laboratory were cultured in DMEM containing 5% fetal bovine serum at 37°C in a 5% CO2 incubator and passaged to the third generation. Cells were then seeded at 5 × 10⁶ cells per well. 5Cells were evenly seeded in 6-well plates at a uniform concentration and cultured until the logarithmic growth phase. After approximately 16 hours, when the cells reached about 70%–75% of the volume per well, the DMEM solution containing 5% fetal bovine serum was discarded. The cells were washed three times with PBS buffer, with the last wash removing all remaining PBS. 1 mL of empty DMEM and PRVXJ5 (MOI = 0.1) were added and incubated with the cells for 1 hour. After 1 hour, the solution was replaced with 2 mL of DMEM containing 2% fetal bovine serum, and PBS buffer or appropriate concentrations of Rehmannia glutinosa polysaccharide (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL) were added. Cell morphology and pathological changes were observed under a microscope at 12 hours and 24 hours, respectively. Figure 1 The results showed that Rehmannia glutinosa polysaccharide could reduce cytopathic effects induced by PRVXJ5. This indicates that Rehmannia glutinosa polysaccharide can reduce infection by variant porcine pseudorabies virus XJ5.

[0035] Example 3: The inhibitory activity of Rehmannia glutinosa polysaccharide on infection of porcine pseudorabies virus variant strain XJ5 in PK-15 cells.

[0036] (1) Western blot analysis of the inhibitory activity of Rehmannia glutinosa polysaccharide on the infection of porcine pseudorabies virus variant XJ5 in PK-15 cells:

[0037] PK-15 cells were digested with trypsin and then diluted with DMEM nutrient solution containing 5% fetal bovine serum at a concentration of 5 × 10⁻⁶ mcg. 5 The concentration of the solution was added dropwise to 6-well plates and incubated at 37°C with 5% CO2 until the cells adhered to the wall and entered the logarithmic growth phase (approximately 17 hours). The cells were then washed three times with PBS buffer, and residual PBS was aspirated. The porcine pseudorabies virus variant XJ5 was added and incubated with PK-15 cells at 37°C with 5% CO2 for 1 hour. The solution was then replaced with 2 mL of DMEM containing 2% fetal bovine serum and appropriate concentrations of Rehmannia glutinosa polysaccharides (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL). The cells were incubated at 37°C with 5% CO2 for 24 hours after infection. The cell supernatant (1 mL) was collected and stored at -70°C for later TCID treatment. 50 (For experimental preparation), wash three times with PBS buffer, aspirate all residual liquid, add 2× protein loading solution to collect cell samples, boil in a metal bath at 96℃ for 15 min, and perform Western blot analysis. It was found that Rehmannia glutinosa polysaccharide reduced the expression of porcine pseudorabies virus gB protein, such as... Figure 2 As shown in A, Figure 2 B is Figure 2 Results A: Gray-scale scanning analysis of gB protein preliminarily confirmed that Rehmannia glutinosa polysaccharide reduces the infection of the pseudorabies virus variant XJ5 in pigs.

[0038] (2)TCID50 The activity of Rehmannia glutinosa polysaccharide in inhibiting infection of porcine pseudorabies virus variant XJ5 in PK-15 cells was determined:

[0039] Vero cells were digested with trypsin and then diluted with DMEM nutrient solution containing 8% fetal bovine serum at a concentration of 2 × 10⁻⁶ mcg. 3 The concentration of virus solution was added to each well of a 96-well plate and incubated at 37°C with 5% CO2 until cells adhered to the plate and entered the logarithmic growth phase (approximately 16 hours). After washing three times with PBS buffer and aspirating any residual liquid, different concentrations of virus solution diluted with serum-free DMEM were added, with four replicates for each concentration. After 1.5 hours of infection, the plate was maintained with DMEM containing 2% fetal bovine serum. Cytopathic effects were observed 72 hours after infection. It was found that Rehmannia glutinosa polysaccharide reduced the viral titer in the supernatant of the porcine pseudorabies virus variant XJ5. Figure 2 As shown in Figure C, Rehmannia glutinosa polysaccharide reduces the infection of the porcine pseudorabies virus variant XJ5.

[0040] (3) Indirect Immunofluorescence Assay (IFA) was used to determine the activity of Rehmannia glutinosa polysaccharide in inhibiting the infection of porcine pseudorabies virus variant XJ5 on PK-15 cells:

[0041] PK-15 cells were digested with trypsin and then diluted with DMEM nutrient solution containing 5% fetal bovine serum at a concentration of 5 × 10⁻⁶ mcg. 5The concentration of the solution was added dropwise to a 6-well plate and incubated at 37°C with 5% CO2 until the cells adhered to the wall and entered the logarithmic growth phase (approximately 17 hours). The cells were then washed three times with PBS buffer, and residual PBS was aspirated. A variant of porcine pseudorabies virus, XJ5, was added and incubated with PK-15 cells at 37°C with 5% CO2 for 1 hour. The solution was then replaced with 2 mL of DMEM containing 2% fetal bovine serum and appropriate concentrations of Rehmannia glutinosa polysaccharides (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL). The cells were incubated at 37°C with 5% CO2 for 24 hours after infection. The supernatant was discarded, and the cells were washed three times with PBS buffer, with residual PBS aspirated on the last wash. A solution sufficient to cover the cells was then added. Cells were fixed with 4% paraformaldehyde at 37°C for 15 minutes, then penetrated with 0.1% Triton X-100 for 10 minutes. After washing three times with PBS buffer, the cells were aspirated thoroughly after the last wash. 5% BSA blocking buffer was added, and the cells were incubated at 37°C for 1.5 hours or overnight at 4°C. After washing three times with PBS, the cells were aspirated thoroughly after the last wash. The cells were then incubated with a 1:200 dilution of the primary antibody PRV porcine positive serum at 37°C for 2 hours. After washing three times with PBST, the cells were aspirated thoroughly after the last wash. The cells were then incubated with a 1:200 dilution of the secondary antibody goat anti-porcine IgG at 37°C for 1 hour in the dark. After staining with DAPI for 5 minutes in the dark, the cells were washed three times with PBST, the cells aspirated thoroughly after the last wash. Cell morphology and lesions were observed under a fluorescence microscope. Figure 2 As shown in Figure D, Rehmannia glutinosa polysaccharide reduced the infection of the porcine pseudorabies virus variant XJ5.

[0042] Example 3: Rehmannia glutinosa polysaccharide inhibits the adsorption and entry of porcine pseudorabies virus variant strain XJ5 into PK-15 cells.

[0043] (1) Western blot detection of the inhibitory effect of Rehmannia glutinosa polysaccharide on the adsorption and entry of porcine pseudorabies virus mutant strain XJ5 on PK-15 cells:

[0044] PK-15 cells were digested with trypsin and then diluted with DMEM nutrient solution containing 5% fetal bovine serum at a concentration of 5 × 10⁻⁶ mcg. 5The concentration of DMEM was added to each well of a 6-well plate and incubated at 37°C with 5% CO2 until the cells adhered to the plate and entered the logarithmic growth phase (approximately 17 hours). The cells were then washed three times with 4°C PBS buffer. After aspirating the residual PBS, 1 mL of serum-free 4°C DMEM and 0.1 MOI of the porcine pseudorabies virus variant XJ5 were added and incubated with PK-15 cells at 4°C for 1 hour. The solution was then replaced with 2 mL of DMEM containing 2% fetal bovine serum and incubated at 37°C with 5% CO2 for 1 hour, with appropriate concentrations of Rehmannia glutinosa polysaccharides (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL). The cells were then cultured at 37°C with 5% CO2, washed three times with citric acid, and then three times with PBS buffer. After aspirating the residual liquid, the solution was replaced with DMEM containing 2% fetal bovine serum. 24 hours after infection, the cell supernatant (1 mL) was collected and stored at -70°C for later TCID45 incubation. 50 (For experimental preparation), cells were washed three times with PBS buffer, and residual liquid was aspirated. 2× protein loading was added to collect cell samples, which were then boiled in a metal bath at 96℃ for 15 min before Western blot analysis. It was found that Rehmannia glutinosa polysaccharide reduced the expression of the XJ5gB protein in the porcine pseudorabies virus variant. Figure 3 As shown in Figures A and B, Rehmannia glutinosa polysaccharide reduced the adsorption and cell entry of the pseudorabies virus variant XJ5.

[0045] (2) TCID50 assay showed that Rehmannia glutinosa polysaccharide inhibited the adsorption and cell entry of porcine pseudorabies virus mutant strain XJ5 on PK-15 cells:

[0046] Vero cells were digested with trypsin and then diluted with DMEM nutrient solution containing 8% fetal bovine serum at a concentration of 2 × 10⁻⁶ mcg. 3 The concentration of virus solution was added dropwise to 96-well plates and incubated at 37°C with 5% CO2 until cells adhered to the plate and entered the logarithmic growth phase (approximately 16 hours). Cells were then washed three times with PBS buffer, and residual liquid was aspirated. Different concentrations of virus solution diluted with serum-free DMEM were added, with four replicates for each concentration. After 1.5 hours of infection, the solution was replaced with DMEM containing 2% fetal bovine serum for maintenance. Cytopathic effects were observed 72 hours after infection. It was found that Rehmannia glutinosa polysaccharide reduced the viral titer in the supernatant of the porcine pseudorabies virus variant XJ5. Figure 3 As shown in Figure C, Rehmannia glutinosa polysaccharide reduced the adsorption and cell entry of the pseudorabies virus variant XJ5.

[0047] (3) Indirect Immunofluorescence Assay (IFA) determination of the inhibitory effect of Rehmannia glutinosa polysaccharide on the adsorption and cell entry of porcine pseudorabies virus variant strain XJ5 on PK-15 cells:

[0048] PK-15 cells were digested with trypsin and then diluted with DMEM nutrient solution containing 5% fetal bovine serum at a concentration of 5 × 10⁻⁶ mcg. 5 The concentration of DMEM was added to each well of a 6-well plate and incubated at 37°C with 5% CO2 until the cells adhered to the plate and entered the logarithmic growth phase (approximately 17 hours). The cells were then washed three times with 4°C PBS buffer. After aspirating the residual PBS, 1 mL of serum-free 4°C DMEM and 0.1 MOI of the porcine pseudorabies virus variant XJ5 were added and incubated with PK-15 cells at 4°C for 1 hour. The solution was then replaced with 2 mL of DMEM containing 2% fetal bovine serum and incubated at 37°C with 5% CO2 for 1 hour, with appropriate concentrations of Rehmannia glutinosa polysaccharides (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL). The cells were then cultured at 37°C with 5% CO2, washed three times with citric acid, and then three times with PBS buffer. After aspirating the residual liquid, DMEM containing 2% fetal bovine serum was added. After 24 hours of infection, the supernatant was discarded. Wash three times with PBS buffer, aspirating the last residual PBS buffer. Add 4% paraformaldehyde (enough to cover the cell surface) and fix at 37°C for 15 minutes. Then, penetrate the cells with 0.1% Triton X-100 for 10 minutes. Wash three more times with PBS buffer, aspirating the last residue. Add 5% BSA blocking solution and incubate at 37°C for 1.5 hours or 4°C overnight. Wash three times with PBS buffer, aspirating the last residue. Incubate with the primary antibody PRV porcine positive serum (1:200 dilution) at 37°C for 2 hours. Wash three times with PBST, aspirating the last residue. Incubate with the secondary antibody goat anti-porcine IgG (1:200 dilution) at 37°C for 1 hour in the dark. Stain with DAPI for 5 minutes in the dark. Wash three times with PBST, aspirating the last residue. Observe cell morphology and lesions under a fluorescence microscope. Figure 3 As shown in Figure D, Rehmannia glutinosa polysaccharide reduced the adsorption and cell entry of the pseudorabies virus variant XJ5.

[0049] Example 4: Rehmannia glutinosa polysaccharide inhibits the adsorption of porcine pseudorabies virus variant strain XJ5 on PK-15 cells.

[0050] (1) Western blot detection of the inhibitory effect of Rehmannia glutinosa polysaccharide on the adsorption of porcine pseudorabies virus variant XJ5 on PK-15 cells:

[0051] PK-15 cells were digested with trypsin and then diluted with DMEM nutrient solution containing 5% fetal bovine serum at a concentration of 5 × 10⁻⁶ mcg. 5The concentration of DMEM was added to each well of a 6-well plate and incubated at 37°C with 5% CO2. After the cells adhered to the plate and entered the logarithmic growth phase (approximately 17 hours), the cells were washed three times with 4°C PBS buffer. After removing the residual PBS, 1 mL of serum-free 4°C DMEM and 0.1 MOI of the porcine pseudorabies virus variant XJ5 were added and incubated with PK-15 cells at 4°C for 1 hour. Then, the solution was replaced with 2 mL of DMEM containing 2% fetal bovine serum and incubated at 37°C with 5% CO2. After 24 hours of infection, the cell supernatant (1 mL) was collected and stored at -70°C as late-stage TCID. 50 (For experimental preparation), cells were washed three times with PBS buffer, and residual liquid was aspirated. Cell samples were collected by adding 2× protein loading solution and boiled in a metal bath at 96℃ for 15 min before Western blot analysis. It was found that Rehmannia glutinosa polysaccharide reduced the expression of the XJ5gB protein in the porcine pseudorabies virus variant. Figure 4 As shown in Figures A and B, it is confirmed that Rehmannia glutinosa polysaccharide reduces the adsorption of the pseudorabies virus variant strain XJ5 in pigs.

[0052] (2)TCID 50 Determination of the inhibitory effect of Rehmannia glutinosa polysaccharide on the adsorption of porcine pseudorabies virus variant XJ5 on PK-15 cells:

[0053] Vero cells were digested with trypsin and then diluted with DMEM nutrient solution containing 8% fetal bovine serum at a concentration of 2 × 10⁻⁶ mcg. 3 The concentration of virus solution was added dropwise to 96-well plates and incubated at 37°C with 5% CO2 until cells adhered to the plate and entered the logarithmic growth phase (approximately 16 hours). Cells were then washed three times with PBS buffer, and residual liquid was aspirated. Different concentrations of virus solution diluted with serum-free DMEM were added, with four replicates for each concentration. After 1.5 hours of infection, the solution was replaced with DMEM containing 2% fetal bovine serum for maintenance. Cytopathic effects were observed 72 hours after infection. It was found that Rehmannia glutinosa polysaccharide reduced the viral titer in the supernatant of the porcine pseudorabies virus variant XJ5. Figure 4 As shown in Figure C, Rehmannia glutinosa polysaccharide reduced the adsorption of the pseudorabies virus variant XJ5 in pigs.

[0054] (3) Indirect Immunofluorescence Assay (IFA) determination of the inhibitory effect of Rehmannia glutinosa polysaccharide on the adsorption of porcine pseudorabies virus variant strain XJ5 on PK-15 cells:

[0055] PK-15 cells were digested with trypsin and then diluted with DMEM nutrient solution containing 5% fetal bovine serum at a concentration of 5 × 10⁻⁶ mcg. 5The concentration of DMEM was added dropwise to 6-well plates and incubated at 37°C with 5% CO2 until the cells adhered to the wall and entered the logarithmic growth phase (approximately 17 hours). The cells were then washed three times with 4°C PBS buffer. After removing the residual PBS, 1 mL of serum-free 4°C DMEM and 0.1 MOI of the porcine pseudorabies virus variant XJ5 were added and incubated with PK-15 cells at 4°C for 1 hour. The solution was then replaced with 2 mL of DMEM containing 2% fetal bovine serum and incubated at 37°C with 5% CO2. After 24 hours of infection, the cells were washed three times with PBS buffer, and the residual solution was removed. 4% paraformaldehyde (enough to cover the cell surface) was added and the cells were fixed at 37°C for 15 minutes. Subsequently, the cells were... 0.1% Triton X-100 penetrated the cells for 10 minutes, then washed three times with PBS buffer, aspirating the last wash thoroughly. Add 5% BSA blocking solution and incubate at 37°C for 1.5 hours or 4°C overnight. Wash three times with PBS buffer, aspirating the last wash thoroughly. Incubate with the primary antibody PRV porcine positive serum (1:200 dilution) at 37°C for 2 hours. Wash three times with PBST, aspirating the last wash thoroughly. Incubate with the secondary antibody goat anti-porcine IgG (1:200 dilution) at 37°C for 1 hour in the dark. Stain with DAPI for 5 minutes in the dark. Wash three times with PBST, aspirating the last wash thoroughly. Observe cell morphology and lesions under a fluorescence microscope. Figure 4 As shown in Figure D, Rehmannia glutinosa polysaccharide reduced the adsorption of the pseudorabies virus variant XJ5 in pigs.

[0056] Example 5: Effects of Rehmannia glutinosa polysaccharide on the cell entry of porcine pseudorabies virus variant strain XJ5 in PK-15 cells

[0057] (1) Western blot detection of the effect of Rehmannia glutinosa polysaccharide on the cell entry of porcine pseudorabies virus variant XJ5 on PK-15 cells:

[0058] PK-15 cells were digested with trypsin and then diluted with DMEM nutrient solution containing 5% fetal bovine serum at a concentration of 5 × 10⁻⁶ mcg. 5The concentration of DMEM was added to each well of a 6-well plate and incubated at 37°C with 5% CO2. After the cells adhered to the plate and entered the logarithmic growth phase (approximately 17 hours), the cells were washed three times with 4°C PBS buffer. After aspirating the residual PBS buffer, 1 mL of serum-free 4°C DMEM and the porcine pseudorabies virus variant XJ5 were added and incubated with PK-15 cells at 4°C for 1 hour. After washing three times with PBS, 1 mL of serum-free DMEM diluted to the appropriate concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL) was added and incubated with PK-15 cells at 37°C with 5% CO2 for 1 hour. After washing three times with citric acid and three times with PBS buffer, the residual solution was aspirated, and the solution was replaced with 2 mL of DMEM containing 2% fetal bovine serum. The plate was then incubated at 37°C with 5% CO2. After 24 hours of infection, the cell supernatant (1 mL) was collected and stored at -70°C for later TCID45 incubation. 50 (For experimental preparation), add 2× protein loading to collect cell samples, boil in a metal bath at 96℃ for 15 min, and perform Western blot analysis. It was found that Rehmannia glutinosa polysaccharide affects the expression of the XJ5gB protein in the porcine pseudorabies virus variant. Figure 5 As shown in A, and for Figure 5 The results obtained from A are subjected to grayscale analysis, such as... Figure 5 As shown in Figure B, it was confirmed that Rehmannia glutinosa polysaccharide at concentrations of 200 μg / ml and 400 μg / ml significantly affected the cell entry of the porcine pseudorabies virus variant XJ5.

[0059] (2)TCID 50 Determination of the effect of Rehmannia glutinosa polysaccharide on the cell entry of porcine pseudorabies virus variant XJ5 on PK-15 cells:

[0060] Vero cells were digested with trypsin and then diluted with DMEM nutrient solution containing 8% fetal bovine serum at a concentration of 2 × 10⁻⁶ mcg. 3 The concentration of virus solution was added to each well of a 96-well plate and incubated at 37°C with 5% CO2 until the cells adhered to the plate and entered the logarithmic growth phase (approximately 16 hours). The cells were then washed three times with PBS buffer, and residual liquid was aspirated. Different concentrations of virus solution diluted with serum-free DMEM were added, with four replicates for each concentration. After 1.5 hours of infection, the solution was replaced with DMEM containing 2% fetal bovine serum for maintenance. Cytopathic effects were observed 72 hours after infection. It was found that Rehmannia glutinosa polysaccharide affected the viral titer in the supernatant of the porcine pseudorabies virus variant XJ5. Figure 5 As shown in Figure C, it is confirmed that Rehmannia glutinosa polysaccharide affects the cell entry of the porcine pseudorabies virus variant XJ5.

[0061] (3) Indirect Immunofluorescence Assay (IFA) showed that Rehmannia glutinosa polysaccharide did not significantly affect the cell entry of the porcine pseudorabies virus variant XJ5 into PK-15 cells:

[0062] PK-15 cells were digested with trypsin and then diluted with DMEM nutrient solution containing 10% fetal bovine serum at a concentration of 5 × 10⁻⁶ mcg. 5 The concentration of DMEM was added dropwise to 6-well plates and incubated at 37°C with 5% CO2 until the cells adhered to the wall and entered the logarithmic growth phase (approximately 17 hours). The cells were then washed three times with 4°C PBS buffer. After aspirating the residual PBS buffer, 1 mL of serum-free 4°C DMEM and the porcine pseudorabies virus variant XJ5 were added and incubated with PK-15 cells at 4°C for 1 hour. The cells were then washed three times with PBS. 1 mL of serum-free DMEM diluted to the appropriate concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL) was added and incubated with PK-15 cells at 37°C with 5% CO2 for 1 hour. The cells were then washed three times with citric acid and three times with PBS buffer. After aspirating the residual solution, 2 mL of DMEM containing 2% fetal bovine serum was added and incubated at 37°C with 5% CO2. The cells were then discarded after 24 hours of infection. The supernatant was washed three times with PBS buffer, with the last wash removing any residual PBS buffer. 4% paraformaldehyde (enough to cover the cell surface) was added and the cells were fixed at 37°C for 15 minutes. Then, 0.1% Triton X-100 was used to penetrate the cells for 10 minutes. The cells were washed three times with PBS buffer, with the last wash removing any remaining PBS buffer. 5% BSA blocking buffer was added and the cells were incubated at 37°C for 1.5 hours or overnight at 4°C. The cells were then washed three times with PBS buffer, with the last wash removing any remaining PBS buffer. The cells were then incubated with a 1:200 dilution of the primary antibody PRV porcine positive serum at 37°C for 2 hours. The cells were washed three times with PBST, with the last wash removing any remaining PBS buffer. The cells were then incubated at 37°C for 1 hour in the dark with a 1:200 dilution of the secondary antibody goat anti-porcine IgG. DAPI staining was performed for 5 minutes in the dark. The cells were washed three times with PBST, with the last wash removing any remaining PBS buffer. Cell morphology and pathological changes were observed under a fluorescence microscope. Figure 5 As shown in Figure D, it is confirmed that Rehmannia glutinosa polysaccharide does not significantly affect the cell entry of the porcine pseudorabies virus variant XJ5.

[0063] Example 6: Rehmannia glutinosa polysaccharide affects the replication of porcine pseudorabies virus variant strain XJ5.

[0064] PK-15 cells were digested with trypsin and then diluted with DMEM nutrient solution containing 5% fetal bovine serum at a concentration of 5 × 10⁻⁶ mcg. 5The concentration of DMEM was added to each well of a 6-well plate and incubated at 37°C with 5% CO2 until the cells adhered to the plate and entered the logarithmic growth phase (approximately 17 hours). The cells were then washed three times with PBS buffer, and any residual PBS buffer was aspirated. 1 mL of serum-free DMEM containing the porcine pseudorabies virus variant XJ5 was added and incubated at 37°C with 5% CO2 for 1 hour. The cells were then washed three times with PBS buffer, and any residual buffer was aspirated. 2 mL of DMEM containing 2% fetal bovine serum and appropriate concentrations of Rehmannia glutinosa polysaccharides (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL) was added. Cell samples were collected at 4 and 6 hours post-infection and then analyzed by Western blot. The results were analyzed using grayscale. The results are as follows: Figure 6 As shown, Rehmannia glutinosa polysaccharide was found to affect the expression of porcine pseudorabies virus gB protein, confirming that Rehmannia glutinosa polysaccharide affects the replication of the porcine pseudorabies virus variant XJ5.

[0065] Example 7:

[0066] Western blot analysis showed that Rehmannia glutinosa polysaccharides may reduce oxidative stress, such as the production of reactive oxygen species (ROS), and decrease the infection rate of the porcine pseudorabies virus variant XJ5 in PK-15 cells.

[0067] PK-15 cells were digested with trypsin and then diluted with DMEM nutrient solution containing 5% fetal bovine serum at a concentration of 5 × 10⁻⁶ mcg. 5 The concentration of DMEM was added to each well of a 6-well plate and incubated at 37°C with 5% CO2 until the cells adhered to the plate and entered the logarithmic growth phase (approximately 17 hours). The cells were then washed three times with PBS buffer. After removing any residual PBS buffer, 1 mL of empty DMEM was added, and the plate was infected with 0.1 MOI of the porcine pseudorabies virus variant XJ5. The plate was incubated at 37°C with 5% CO2 for 1 hour. The incubator was then replaced with 2 mL of DMEM containing 2% fetal bovine serum and appropriate concentrations of Rehmannia glutinosa (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL). After 24 hours of infection, the cells were stained with DCFH-DA, digested with trypsin, and then collected for flow cytometry analysis. Results are as follows: Figure 7 As shown, Rehmannia glutinosa polysaccharide inhibited the production of reactive oxygen species (ROS) induced by porcine pseudorabies virus (PRV), and reduced the infection effect of the PRV variant XJ5 strain, confirming that Rehmannia glutinosa polysaccharide may reduce oxidative stress and play a therapeutic role.

[0068] Example 8:

[0069] CCK8-Kit assay for the cytotoxic effect of Rehmannia glutinosa on PK-15 cells:

[0070] When PK-15 cells reached a density of 70%–80%, they were digested with 0.25% trypsin, followed by termination of digestion with 5% DMEM. Approximately 5000 cells per well (100 mL) were cultured in 96-well plates. After about 17 hours, different concentrations of Rehmannia glutinosa polysaccharide (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL) were added to each well, with four replicates for each concentration. PBS was used as a control group. After culturing at 37°C and 5% CO2 for 24 hours, 10 mL of CK-8 reagent was added to each well, and the plates were incubated at 37°C and 5% CO2 for 2 hours. The absorbance was measured at 450 nm using a microplate reader. The results are as follows: Figure 8 As shown, this confirms that Rehmannia glutinosa polysaccharide has no toxic effect on PK-15 cells.

[0071] Based on the toxicity and related properties of Rehmannia glutinosa polysaccharide, 400 μg / mL is the preferred dosage.

[0072] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of Rehmannia glutinosa polysaccharide in the preparation of an anti-pseudorabies virus preparation for the prevention and / or treatment of porcine pseudorabies, wherein the concentration of Rehmannia glutinosa polysaccharide in the anti-pseudorabies virus preparation is 50-400 μg / mL; wherein the anti-pseudorabies virus preparation prevents and / or treats porcine pseudorabies by inhibiting the adsorption of porcine pseudorabies virus; The anti-pseudorabies virus infection agent prevents and / or treats swine pseudorabies by inhibiting the entry of swine pseudorabies virus into cells; The anti-pseudorabies virus infection preparation prevents and / or treats pseudorabies by inhibiting the replication of the pseudorabies virus.

2. The application as described in claim 1, characterized in that, The concentration of Rehmannia glutinosa polysaccharide in the anti-swine pseudorabies virus preparation is 400 μg / mL.

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