CN113171374B

CN113171374B - Application of sea buckthorn polysaccharide in preparation of preparation for preventing and/or treating porcine pseudorabies virus infection

Info

Publication number: CN113171374B
Application number: CN202110643760.4A
Authority: CN
Inventors: 郇长超; 张伟; 刘金彪; 尼博; 高崧
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-06-09
Filing date: 2021-06-09
Publication date: 2022-04-15
Anticipated expiration: 2041-06-09
Also published as: CN113171374A

Abstract

The invention discloses application of sea buckthorn polysaccharide in preparation of a preparation for preventing and/or treating porcine pseudorabies virus infection. In vitro cell experiments prove that the hippophae rhamnoides polysaccharide can effectively inhibit infection of the porcine pseudorabies virus and can remarkably reduce cytopathic effect caused by the porcine pseudorabies virus. The addition of the hippophae rhamnoides polysaccharide at different stages of virus infection is found to mainly inhibit the adsorption, cell entering and replication of viruses on cells. The invention provides a safe and effective preparation with small toxic and side effects for clinically preventing and/or treating the porcine pseudorabies virus infection.

Description

Application of sea buckthorn polysaccharide in preparation of preparation for preventing and/or treating porcine pseudorabies virus infection

Technical Field

The invention belongs to the field of antiviral application, and particularly relates to application of sea buckthorn polysaccharide in preparation of a preparation for preventing and/or treating porcine pseudorabies virus infection.

Background

Pseudorabies (PR), also known as Aujeszky's Disease (AD), is an acute and virulent infectious disease caused by Pseudorabies virus (PRV), and mainly causes symptoms such as fever, pruritus and encephalomyelitis of various domestic animals and wild animals. PRV is a double-stranded DNA virus belonging to the sub-family herpesviridae and herpesviridae. While PRV can infect a variety of mammals, such as dogs, cats, cattle, sheep and rabbits, pigs are the only natural host. The porcine pseudorabies is one of important diseases harming the pig industry, pigs of all ages and sexes are easy to be infected with the porcine pseudorabies virus, the infection of newborn piglets can cause 100 percent of death rate, and huge economic loss is caused to the world pig industry. For this reason, the world animal health Organization (OIE) lists it as an animal disease to be notified.

In China, the genetic engineering deletion vaccine is mainly used for carrying out immunization (such as the gE deletion vaccine) on the pigs before 2011, so that the disease condition is well controlled, and the disease condition in a large range is not generated. However, in 2011, new conditions such as mutation, virulence enhancement and the like of PRV occur, which leads to the outbreak of epidemic situation in many pig farms. Furthermore, there is increasing evidence that the classical Bartha-K61 vaccine does not completely protect piglets from infection by these mutant viruses. More seriously, china reported the first endophthalmitis cases caused by PRV infection in 2017, and recently isolated PRV from an acute human encephalitis case, indicating that the new mutant strain has been able to infect humans. Thus, prevention and control of PRV infection is particularly acute.

Currently, the long-term application of chemically synthesized antiviral drugs causes a number of problems, such as drug resistance, drug residues, environmental pollution of drug metabolites, and the like. Therefore, the method has very important significance in searching and developing antiviral drugs with high efficiency, low toxicity and strong specificity. The traditional Chinese medicine is a treasure in the traditional Chinese culture, has thousands of years of clinical experience, and makes great contribution to human prevention and treatment of diseases and health maintenance. In recent years, the research on Chinese medicine antiviral at home and abroad is gradually increased, and the attempt to screen antiviral active ingredients from Chinese medicine treasury becomes a hot spot of the current new medicine research and development.

Sea-buckthorn (Hippophae rhamnoides L.) is a traditional medicinal plant in China and has the effects of enhancing immunity, resisting aging, preventing vascular embolism and the like. Polysaccharides are essential substances for organisms and are closely related to maintaining biological functions, and natural polysaccharides have various biological functions such as oxidation resistance, antibiosis, antivirus and the like. Researches find that the sea buckthorn polysaccharide has the functions of resisting oxidation, regulating immunity, resisting tumors and the like, has high medicinal value and bioactivity and has wide development prospect. However, the inhibitory effect of Hippophae Rhamnoides Polysaccharides (HRP) on viruses has not been reported.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the application of the hippophae rhamnoides polysaccharide in preparing the preparation for preventing and/or treating the porcine pseudorabies virus infection, and provides a safe and effective preparation with small toxic and side effects for clinically preventing and/or treating the porcine pseudorabies virus infection.

In some embodiments of the invention, the hippophae rhamnoides polysaccharide is capable of reducing cytopathic effects caused by porcine pseudorabies virus; and/or

The sea buckthorn polysaccharide can reduce the expression of porcine pseudorabies virus gB protein; and/or

The sea buckthorn polysaccharide can reduce the virus titer of the porcine pseudorabies virus infection; and/or

The sea buckthorn polysaccharide can reduce the copy number of nucleic acid of the porcine pseudorabies virus.

In some embodiments of the invention, the hippophae rhamnoides polysaccharide is capable of inhibiting porcine pseudorabies virus adsorption; and/or

The sea buckthorn polysaccharide can inhibit the entrance of the porcine pseudorabies virus; and/or

The sea buckthorn polysaccharide can inhibit the replication of the porcine pseudorabies virus.

In the invention, the sea buckthorn polysaccharide plays a role in resisting viruses by inhibiting the absorption, cell entering and virus replication of the porcine pseudorabies virus.

In some embodiments of the invention, the porcine pseudorabies virus further comprises a variant porcine pseudorabies virus.

In some preferred embodiments of the invention, the strain of the variant porcine pseudorabies virus is the PRV XJ5 strain.

In some embodiments of the invention, the formulation comprises a pharmaceutical formulation, a disinfectant or a feed additive. Namely, the method comprises the following steps: the application of the sea buckthorn polysaccharide in preparing the medicine for preventing and/or treating the infection of the porcine pseudorabies virus; and/or

The application of the sea buckthorn polysaccharide in preparing disinfectant for preventing and/or treating porcine pseudorabies virus infection; and/or

Application of sea buckthorn polysaccharide in preparing feed additive for preventing and/or treating porcine pseudorabies virus infection.

In some preferred embodiments of the present invention, the pharmaceutical formulation further comprises a pharmaceutically acceptable salt and/or an adjuvant.

Further, the pharmaceutically acceptable carrier refers to a pharmaceutical carrier conventional in the pharmaceutical field, such as: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite clay; lubricants such as talc, calcium stearate and magnesium stearate, and polyethylene glycol, and the like. Other adjuvants such as sweetener, flavoring agent, etc. can also be added into the composition.

Further, the pharmaceutical preparation is prepared into any pharmaceutically acceptable preparation according to the requirement, including tablets, capsules, granules, injections, pills, syrups, powders or ointments.

In some embodiments of the invention, the concentration of the hippophae rhamnoides polysaccharide is 250 to 1000. mu.g/mL.

In some preferred embodiments of the invention, the concentration of the hippophae rhamnoides polysaccharide is 500 to 1000. mu.g/mL.

In some more preferred embodiments of the invention, the concentration of the hippophae rhamnoides polysaccharide is 750 to 1000. mu.g/mL.

In some embodiments of the invention, the formulation is prepared by dissolving the hippophae rhamnoides polysaccharide in a solvent.

In some preferred embodiments of the invention, the solvent is PBS buffer.

The application of the invention has at least the following beneficial effects: the invention provides a new application of seabuckthorn polysaccharide, namely the application of seabuckthorn polysaccharide in preparing a preparation for preventing and/or treating PRV infection, so as to provide a new way for clinically preventing and treating PRV infection; cell infection tests prove that the seabuckthorn polysaccharide can effectively inhibit PRV infection, can reduce cytopathy caused by PRV infection, and inhibit adsorption and entry of PRV to cells, thereby providing scientific and reliable theoretical basis for clinical treatment of porcine pseudorabies; compared with the traditional chemical antiviral drugs, the hippophae rhamnoides polysaccharide is a natural product with high safety and small preparation pollution, and has the advantages of higher safety and effectiveness, capability of resisting variant PRV and the like.

The sea-buckthorn polysaccharide is deciduous shrubs, can resist drought and wind sand, and can survive on saline-alkali soil, so that the sea-buckthorn polysaccharide is widely applied to water and soil conservation in China, and has the advantages of wide medicine source, readily available raw materials, low cost and the like; the sea buckthorn fruit is edible, so that the sea buckthorn fruit is non-toxic and harmless to animals, and has no pollution to the environment after being metabolized by the animals; studies have found that the sea buckthorn polysaccharide has various biological activities, such as antioxidation, liver protection, blood sugar regulation, cholesterol reduction and the like.

In the present invention, the term "treating" includes alleviating, inhibiting or ameliorating the symptoms or conditions of a disease; inhibiting the generation of complications: ameliorating or preventing underlying metabolic syndrome; inhibiting the development of a disease or condition, such as controlling the development of a disease or condition; alleviating the disease or symptoms; regression of the disease or symptoms; alleviating a complication caused by the disease or symptom, or preventing or treating a symptom caused by the disease or symptom. As used herein, administration can result in an improvement in a disease, symptom, or condition, particularly an improvement in severity, delay in onset, slow progression, or decrease in duration of a condition.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a western blot result chart of experiments on inhibition of porcine pseudorabies virus infection by seabuckthorn polysaccharide in example 5 of the present invention;

FIG. 2 is a diagram showing an image of cells under a microscope for observing the effect of hippophae rhamnoides polysaccharide on cytopathic effect caused by porcine pseudorabies virus infecting PK-15 cells in example 6 of the present invention;

FIG. 3 is a graph showing the effect of seabuckthorn polysaccharide on PK-15 cells for porcine pseudorabies virus infection in example 7 of the present invention; wherein A is Western blot for determining the influence of the hippophae rhamnoides polysaccharide on PK-15 cells on porcine pseudorabies virus infection; b is TCID₅₀Determining the influence of the sea buckthorn polysaccharide on PK-15 cells on the infection of the porcine pseudorabies virus; c, measuring the influence of the sea buckthorn polysaccharide on PK-15 cells on the infection of the porcine pseudorabies virus by indirect immunofluorescence;

FIG. 4 is a graph showing the effect of seabuckthorn polysaccharide pretreatment of PK-15 cells on porcine pseudorabies virus infection in example 8 of the present invention;

FIG. 5 shows the effect of seabuckthorn polysaccharide on PK-15 cells on porcine pseudorabies virus uptake cells in example 9 of the present invention; wherein A is Western blot for determining the influence of the hippophae rhamnoides polysaccharide on PK-15 cells on porcine pseudorabies virus absorption cells; b is TCID₅₀Determining the influence of the sea buckthorn polysaccharide on PK-15 cells on porcine pseudorabies virus absorption cells; c, measuring the influence of the sea buckthorn polysaccharide on PK-15 cells on porcine pseudorabies virus absorption cells by indirect immunofluorescence;

FIG. 6 shows the effect of seabuckthorn polysaccharide on PK-15 cells for porcine pseudorabies virus adsorption in example 10 of the present invention; wherein A is Western blot for determining the influence of the hippophae rhamnoides polysaccharide on PK-15 cells on porcine pseudorabies virus adsorption; b is TCID₅₀Determining the influence of the sea buckthorn polysaccharide on the PK-15 cells on the adsorption of the porcine pseudorabies virus; c, measuring the influence of the sea buckthorn polysaccharide on PK-15 cells on the adsorption of the porcine pseudorabies virus by indirect immunofluorescence;

FIG. 7 shows the effect of hippophae rhamnoides polysaccharide on PK-15 cells on porcine pseudorabies virus encytosis in example 11 of the present invention; wherein A is Western blot for determining the influence of the hippophae rhamnoides polysaccharide on PK-15 cells on the cell entrance of the porcine pseudorabies virus; b is TCID₅₀Determining the influence of the hippophae rhamnoides polysaccharide on the cell entrance of the porcine pseudorabies virus on PK-15 cells; c, measuring the influence of the sea buckthorn polysaccharide on PK-15 cells on the cell entrance of the porcine pseudorabies virus by indirect immunofluorescence;

FIG. 8 shows a Western blot assay for determining the effect of hippophae rhamnoides polysaccharide on PK-15 cells on porcine pseudorabies virus replication in example 12 of the present invention;

FIG. 9 shows TCID in example 13 of the present invention₅₀The direct effect of the sea buckthorn polysaccharide on killing the porcine pseudorabies virus is measured.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

Example 1

A preparation for preventing and/or treating porcine pseudorabies virus infection is prepared by dissolving fructus Hippophae polysaccharide (Ciyuan Biotechnology Co., Ltd.) in PBS buffer solution at a concentration of 250 μ g/mL.

Example 2

A preparation for preventing and/or treating porcine pseudorabies virus infection is prepared by dissolving fructus Hippophae polysaccharide in PBS buffer solution, wherein the concentration of fructus Hippophae polysaccharide is 500 μ g/mL.

Example 3

A preparation for preventing and/or treating porcine pseudorabies virus infection is prepared by dissolving fructus Hippophae polysaccharide in PBS buffer solution, wherein the concentration of fructus Hippophae polysaccharide is 750 μ g/mL.

Example 4

A preparation for preventing and/or treating porcine pseudorabies virus infection is prepared by dissolving fructus Hippophae polysaccharide in PBS buffer solution, wherein the concentration of fructus Hippophae polysaccharide is 1000 μ g/mL.

The inhibitory effect of Hippophae Rhamnoides Polysaccharides (HRP) on viruses has not been reported. Thus, we investigated the antiviral effect of hippophae rhamnoides polysaccharide in PRV infected host cells in the subsequent examples.

Example 5: sea-buckthorn polysaccharide for inhibiting infection of porcine pseudorabies virus

Cells were pretreated with hippophae rhamnoides polysaccharide (1mg/mL, prepared in example 4) on PK-15 cells for 1 hour, infected with porcine pseudorabies virus (0.1MOI) for 1 hour, with hippophae rhamnoides polysaccharide remaining during infection, and cells were collected at 2h, 4h, 8h, 12h, and 24h for western blot experiments. In this experiment, the control group 1 was prepared without adding polysaccharide and without infecting PRV, and the control group 2 was prepared without adding polysaccharide and without infecting PRV. The obtained western blot experiment result is shown in fig. 1, and the gB protein is expressed after PRV infection is seen, and the gB protein of the cell treated by the seabuckthorn polysaccharide is not expressed, which indicates that the cell treated by the seabuckthorn polysaccharide is not infected by the porcine pseudorabies virus, and the seabuckthorn polysaccharide inhibits the infection of the porcine pseudorabies virus.

Example 6: influence of sea buckthorn polysaccharide on cytopathic effect caused by porcine pseudorabies virus

PK-15 cells were trypsinized, diluted and counted in DMEM containing 4% fetal bovine serum, and the cells were plated in 6-well plates at a cell count of 5X 10 per well⁵At 37 ℃ with 5% CO₂The incubator of (1) overnight. When the cell density reached 70% -80%, the cells were washed 3 times with PBS, serum-free DMEM was added, pretreated for 1h with PBS or hippophae rhamnoides polysaccharide (prepared from examples 1-4 at concentrations of 250. mu.g/mL, 500. mu.g/mL, 750. mu.g/mL, 1000. mu.g/mL, respectively), and then infected with PRV XJ5 strain (0.1 MOI). After 1 hour, cells were washed three times with PBS, replaced with DMEM containing 2% fetal bovine serum to maintain growth, and the corresponding PBS or hippophae rhamnoides polysaccharide (250. mu.g/mL, 500. mu.g/mL, 750. mu.g/mL, 1000. mu.g/mL) was added. At 12 and 24 hours of infection, the cytopathic effect was observed under a microscope. The results are shown in fig. 2, where hippophae rhamnoides polysaccharide reduces cytopathic effects caused by PRV infection. Example 7: activity of sea buckthorn polysaccharide for inhibiting infection of porcine pseudorabies virus (Western blot, IFA and TCID)₅₀)

(1) Western blot determination of activity of hippophae rhamnoides polysaccharide in inhibiting porcine pseudorabies virus infection on PK-15 cells

PK-15 cells were trypsinized, diluted and counted in DMEM containing 4% fetal bovine serum, and the cells were plated in 6-well plates at a cell count of 5X 10 per well⁵At 37 ℃ with 5% CO₂The incubator of (1) overnight. When the cell density reached 70% -80%, the cells were washed 3 times with PBS, serum-free DMEM was added, pretreated with PBS or hippophae rhamnoides polysaccharide (250. mu.g/mL, 500. mu.g/mL, 750. mu.g/mL, 1000. mu.g/mL), respectively, at 37 ℃ for 1h, and then infected with PRV XJ5 strain (0.1 MOI). After 1 hour of infection at 37 ℃, cells were washed three times with PBS, replaced with DMEM containing 2% fetal bovine serum to maintain growth, and the corresponding PBS or sand was addedAcanthan polysaccharide (250. mu.g/mL, 500. mu.g/mL, 750. mu.g/mL, 1000. mu.g/mL). 24 hours after infection, cell supernatants were collected and stored in a-70 ℃ freezer for TCID₅₀The test, the cells with PBS three times washing, adding 2 x protein loading collected cell samples, with 96 degrees C metal bath boiled for 15 minutes, western blot experiment. The results are shown in a in fig. 3, and the hippophae rhamnoides polysaccharide can reduce the expression of porcine pseudorabies virus gB protein, confirming that hippophae rhamnoides polysaccharide can inhibit infection by porcine pseudorabies virus.

(2) IFA (enzyme-linked immunosorbent assay) for determining activity of sea buckthorn polysaccharide on PK-15 cells for inhibiting porcine pseudorabies virus infection

After infection for 24 hours, the cells were fixed with 4% paraformaldehyde in an incubator at 37 ℃ for 30 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and then blocked with 5% BSA at 4 ℃ overnight. After blocking, the cells were incubated with PRV positive pig serum at 37 ℃ for 1h, and then incubated with FITC labeled goat anti-pig IgG antibody at 37 ℃ for 30 min. After these cells were incubated with DAPI for 7min and observed under a fluorescence microscope, all images were taken at 100-fold magnification, and the results are shown in fig. 3C, confirming that hippophae rhamnoides polysaccharide can inhibit infection by porcine pseudorabies virus.

DAPI is 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole), as in the following example.

(3)TCID₅₀Determination of Activity of Hippophae rhamnoides polysaccharide on PK-15 cells for inhibiting infection of porcine pseudorabies virus

Vero cells were trypsinized, diluted and counted in DMEM containing 6% fetal bovine serum at 2X 10³Perwell concentration was added dropwise to 96-well plates at 37 ℃ with 5% CO₂After the cells adhered to the wall to form a monolayer, the cells were washed 3 times with PBS, virus solution diluted with serum-free DMEM was added, each dilution was repeated 8 times, and the cells were incubated at 37 ℃ with 5% CO₂In an incubator. 1.5 hours after infection, cells were washed 3 times with PBS and maintained in DMEM containing 2% fetal bovine serum. Cytopathic effect was observed at 36 hours after infection, and TCID was calculated by Reed-Muench's method₅₀. As a result, as shown in B in FIG. 3, it was found that hippophae rhamnoides polysaccharide can reduce porcine pseudorabiesThe virus titer of the virus infection supernatant proves that the seabuckthorn polysaccharide can inhibit the infection of the porcine pseudorabies virus.

Example 8: influence of Hippophae rhamnoides polysaccharide on infection by porcine pseudorabies virus (Western blot experiment)

PK-15 cells were trypsinized, diluted and counted in DMEM containing 4% fetal bovine serum, and the cells were plated in 6-well plates at a cell count of 5X 10 per well⁵At 37 ℃ with 5% CO₂The incubator of (1) overnight. When the cell density reached 70% -80%, the cells were washed 3 times with PBS, serum-free DMEM was added, pretreated with PBS or hippophae rhamnoides polysaccharide (250. mu.g/mL, 500. mu.g/mL, 750. mu.g/mL, 1000. mu.g/mL), respectively, at 37 ℃ for 1h, then the cells were washed 3 times with PBS, serum-free DMEM was added, and the cells were infected with PRV XJ5 strain (0.1 MOI). After 1 hour of infection at 37 ℃, cells were washed three times with PBS and maintained in growth by changing to DMEM containing 2% fetal bovine serum. After 24 hours of infection, cells were washed three times with PBS, and a cell sample was collected by adding 2X protein loading, and then subjected to western blot after being boiled in a metal bath at 96 ℃ for 15 minutes. The results are shown in FIG. 4, where no effect of HIPPOHA polysaccharide pretreatment on PK-15 cells was observed on infection with porcine pseudorabies virus.

Example 9: effect of sea buckthorn polysaccharide on porcine pseudorabies Virus adsorption and engraftment (Western blot, IFA and TCID)₅₀)

(1) Western blot determination of influence of sea buckthorn polysaccharide on PK-15 cells on porcine pseudorabies virus adsorption and entry cells

PK-15 cells were trypsinized, diluted and counted in DMEM containing 4% fetal bovine serum, and the cells were plated in 6-well plates at a cell count of 5X 10 per well⁵At 37 ℃ with 5% CO₂The incubator of (1) overnight. When the cell density reaches 70-80%, washing the cells for 3 times by PBS, adding cold serum-free DMEM and infecting the porcine pseudorabies virus, wherein seabuckthorn polysaccharide (250 mug/mL, 500 mug/mL, 750 mug/mL, 1000 mug/mL) with corresponding concentration exists in the infection process, after infecting for 1 hour at 4 ℃, washing the cells for three times by PBS, changing into DMEM containing 2% fetal calf serum and adding seabuckthorn polysaccharide (250 mug/mL, 500 mug/mL, 750 mug/mL, 1000 mug/mL) with corresponding concentration, after incubating for 1 hour at 37 ℃,cells were washed 3 times with citrate and then 3 times with PBS, and maintained at 37 ℃ in DMEM containing 2% fetal bovine serum. 24 hours after infection, cell supernatants were collected and stored in a-70 ℃ freezer for TCID₅₀In the test, cells were washed three times with PBS, and 2 Xprotein loading was added to collect cell samples, which were then subjected to a western blot experiment after being boiled in a 96 ℃ metal bath for 15 minutes. The results are shown in a in fig. 5, the hippophae rhamnoides polysaccharide can reduce the expression of porcine pseudorabies virus gB protein, confirming that hippophae rhamnoides polysaccharide can inhibit the adsorption and encytosis of porcine pseudorabies virus.

(2) IFA determination of Effect of Hippophae rhamnoides polysaccharide on PK-15 cells on porcine pseudorabies virus adsorption and engraftment cells were treated in the same manner as above, and 24 hours after infection, the cells were fixed with 4% paraformaldehyde in an incubator at 37 ℃ for 30 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and then blocked with 5% BSA at 4 ℃ overnight. After blocking, the cells were incubated with PRV positive pig serum at 37 ℃ for 1h, and then incubated with FITC labeled goat anti-pig IgG antibody at 37 ℃ for 30 min. After incubation of these cells with DAPI for 7min, all images were taken at 100-fold magnification, observed under a fluorescence microscope. The experimental result is shown as C in figure 5, which proves that the hippophae rhamnoides polysaccharide can inhibit the adsorption and cell entry of the porcine pseudorabies virus.

(3)TCID₅₀Determining the influence of sea buckthorn polysaccharide on PK-15 cells on porcine pseudorabies virus adsorption and entry cells

Vero cells were trypsinized, diluted and counted in DMEM containing 6% fetal bovine serum at 2X 10³Perwell concentration was added dropwise to 96-well plates at 37 ℃ with 5% CO₂After the cells adhered to the wall to form a monolayer, the cells were washed 3 times with PBS, virus solution diluted with serum-free DMEM was added, each dilution was repeated 8 times, and the cells were incubated at 37 ℃ with 5% CO₂In an incubator. 1.5 hours after infection, cells were washed 3 times with PBS and maintained in DMEM containing 2% fetal bovine serum. Cytopathic effect was observed at 36 hours after infection, and TCID was calculated by Reed-Muench's method₅₀. As a result, as shown in B in FIG. 5, it was found that hippophae rhamnoides polysaccharide can reduce the virus titer of supernatant of porcine pseudorabies virus infection, confirming that hippophae rhamnoides polysaccharide can inhibit porcine pseudorabiesAdsorption and entry of canine virus.

Example 10: effect of Hippophae rhamnoides polysaccharide on Swine pseudorabies Virus adsorption (Western blot, IFA and TCID)₅₀)

(1) Western blot determination of influence of sea buckthorn polysaccharide on adsorption of porcine pseudorabies virus on PK-15 cells

PK-15 cells were trypsinized, diluted and counted in DMEM containing 4% fetal bovine serum, and the cells were plated in 6-well plates at a cell count of 5X 10 per well⁵At 37 ℃ with 5% CO₂The incubator of (1) overnight. When the cell density reaches 70% -80%, washing the cells for 3 times by PBS, adding cold serum-free DMEM and infecting the porcine pseudorabies virus, adding sea-buckthorn polysaccharide (250 mug/mL, 500 mug/mL, 750 mug/mL, 1000 mug/mL) with corresponding concentration in the infection process, after infecting for 1 hour at 4 ℃, washing the cells for three times by PBS, and changing the cells into DMEM containing 2% fetal calf serum to maintain at 37 ℃. 24 hours after infection, cell supernatants were collected and stored in a-70 ℃ freezer for TCID₅₀In the test, cells were washed three times with PBS, and 2 Xprotein loading was added to collect cell samples, which were then subjected to a western blot experiment after being boiled in a 96 ℃ metal bath for 15 minutes. The results are shown in a in fig. 6, the hippophae rhamnoides polysaccharide can reduce the expression of porcine pseudorabies virus gB protein, confirming that hippophae rhamnoides polysaccharide can inhibit the adsorption of porcine pseudorabies virus.

(2) IFA determination of influence of sea buckthorn polysaccharide on adsorption of porcine pseudorabies virus on PK-15 cells

After infection for 24 hours, the cells were fixed with 4% paraformaldehyde in an incubator at 37 ℃ for 30 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and then blocked with 5% BSA at 4 ℃ overnight. After blocking, the cells were incubated with PRV positive pig serum at 37 ℃ for 1h, and then incubated with FITC labeled goat anti-pig IgG antibody at 37 ℃ for 30 min. After incubation of these cells with DAPI for 7min, all images were taken at 100-fold magnification, observed under a fluorescence microscope. The experimental result is shown as C in figure 6, which proves that the hippophae rhamnoides polysaccharide can inhibit the adsorption of the porcine pseudorabies virus.

(3)TCID₅₀Determination of the Effect of sea buckthorn polysaccharide on PK-15 cells on porcine pseudorabiesEffect of Virus adsorption

Vero cells were trypsinized, diluted and counted in DMEM containing 6% fetal bovine serum at 2X 10³Perwell concentration was added dropwise to 96-well plates at 37 ℃ with 5% CO₂After the cells adhered to the wall to form a monolayer, the cells were washed 3 times with PBS, virus solution diluted with serum-free DMEM was added, each dilution was repeated 8 times, and the cells were incubated at 37 ℃ with 5% CO₂In an incubator. 1.5 hours after infection, cells were washed 3 times with PBS and maintained in DMEM containing 2% fetal bovine serum. Cytopathic effect was observed at 36 hours after infection, and TCID was calculated by Reed-Muench's method₅₀. The results are shown in fig. 6B, and it was found that hippophae rhamnoides polysaccharide can reduce the virus titer of porcine pseudorabies virus infection supernatant, confirming that hippophae rhamnoides polysaccharide can inhibit the adsorption of porcine pseudorabies virus.

Example 11: effect of Hippophae rhamnoides polysaccharide on porcine pseudorabies Virus encytosis (Westernblot, IFA and TCID)₅₀)

(1) Westernblot for determining influence of hippophae rhamnoides polysaccharide on PK-15 cells on cell entry of porcine pseudorabies virus

PK-15 cells were trypsinized, diluted and counted in DMEM containing 4% fetal bovine serum, and the cells were plated in 6-well plates at a cell count of 5X 10 per well⁵At 37 ℃ with 5% CO₂The incubator of (1) overnight. When the cell density reaches 70% -80%, the cells are washed 3 times by PBS, cold serum-free DMEM is added and the porcine pseudorabies virus is infected, after 1 hour of infection at 4 ℃, the cells are washed 3 times by PBS, DMEM containing 2% fetal calf serum is added, seabuckthorn polysaccharide (250 mu g/mL,500 mu g/mL,750 mu g/mL and 1000 mu g/mL) with corresponding concentration is added, after 1 hour of incubation at 37 ℃, the cells are washed 3 times by citric acid solution, washed 3 times by PBS and then maintained by adding DMEM containing 2% fetal calf serum. 24 hours after infection, cell supernatants were collected and stored in a-70 ℃ freezer for TCID₅₀In the test, cells were washed three times with PBS, and 2 Xprotein loading was added to collect cell samples, which were then subjected to a western blot experiment after being boiled in a 96 ℃ metal bath for 15 minutes. As a result, shown in A in FIG. 7, hippophae rhamnoides polysaccharide was able to reduce the expression of porcine pseudorabies virus gB protein, confirming thatThe fructus Hippophae polysaccharide can inhibit cell entry of porcine pseudorabies virus.

(2) IFA determination of influence of sea buckthorn polysaccharide on porcine pseudorabies virus encytosis on PK-15 cells

After infection for 24 hours, the cells were fixed with 4% paraformaldehyde in an incubator at 37 ℃ for 30 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and then blocked with 5% BSA at 4 ℃ overnight. After blocking, the cells were incubated with PRV positive pig serum at 37 ℃ for 1h, and then incubated with FITC labeled goat anti-pig IgG antibody at 37 ℃ for 30 min. After incubation of these cells with DAPI for 7min, all images were taken at 100-fold magnification, observed under a fluorescence microscope. The experimental result is shown as C in figure 7, which proves that the hippophae rhamnoides polysaccharide can inhibit the adsorption and cell entry of the porcine pseudorabies virus.

(3)TCID₅₀Determining the influence of hippophae rhamnoides polysaccharide on porcine pseudorabies virus encytosis on PK-15 cells

Vero cells were trypsinized, diluted and counted in DMEM containing 6% fetal bovine serum at 2X 10³Perwell concentration was added dropwise to 96-well plates at 37 ℃ with 5% CO₂After the cells adhered to the wall to form a monolayer, the cells were washed 3 times with PBS, virus solution diluted with serum-free DMEM was added, each dilution was repeated 8 times, and the cells were incubated at 37 ℃ with 5% CO₂In an incubator. 1.5 hours after infection, cells were washed 3 times with PBS and maintained in DMEM containing 2% fetal bovine serum. Cytopathic effect was observed at 36 hours after infection, and TCID was calculated by Reed-Muench's method₅₀. The results are shown in fig. 7B, and it was found that hippophae rhamnoides polysaccharide can reduce the virus titer of porcine pseudorabies virus infection supernatant, confirming that hippophae rhamnoides polysaccharide can inhibit the encytosis of porcine pseudorabies virus.

Example 12: effect of sea-buckthorn polysaccharide on replication of porcine pseudorabies virus (Western blot)

PK-15 cells were trypsinized, diluted and counted in DMEM containing 4% fetal bovine serum, and the cells were plated in 6-well plates at a cell count of 5X 10 per well⁵At 37 ℃ with 5% CO₂The incubator of (1) overnight. When the cell density reaches 70-80%The cells were washed 3 times with PBS, serum-free DMEM was added and infected with porcine pseudorabies virus, after infection at 37 ℃ for 1 hour, the cells were washed 3 times with PBS, DMEM containing 2% fetal bovine serum was added to maintain and corresponding concentrations of hippophae rhamnoides polysaccharide (250. mu.g/mL, 500. mu.g/mL, 750. mu.g/mL, 1000. mu.g/mL) were added, the cells were washed three times with PBS at 4 hours and 6 hours of infection, 2 Xprotein loading was added to collect cell samples, and then after cooking for 15 minutes with 96 ℃ metal bath, western blot experiments were performed. The results are shown in fig. 8, and the hippophae rhamnoides polysaccharide reduces the expression level of porcine pseudorabies virus gB protein, and confirms that the hippophae rhamnoides polysaccharide can inhibit the replication of porcine pseudorabies virus.

Example 13: direct killing effect (TCID) of sea buckthorn polysaccharide on porcine pseudorabies virus₅₀)

Incubating PBS or fructus Hippophae polysaccharide solution (250 μ g/mL,500 μ g/mL,750 μ g/mL,1000 μ g/mL) with porcine pseudorabies virus at 37 deg.C for 1 hr, diluting the incubated virus with serum-free virus solution, repeating inoculation of 8 cells in each dilution gradient, inoculating Vero cells in advance, standing at 37 deg.C and 5% CO₂In an incubator. 1.5 hours after infection, cells were washed 3 times with PBS and maintained in DMEM containing 2% fetal bovine serum. Cytopathic effect was observed at 36 hours after infection, and TCID was calculated by Reed-Muench's method₅₀. The results are shown in fig. 9, the virus titer of the virus treated by the hippophae rhamnoides polysaccharide is not reduced, which indicates that the hippophae rhamnoides polysaccharide can not directly kill the porcine pseudorabies virus.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims

1. Application of sea buckthorn polysaccharide in preparing preparation for preventing and/or treating porcine pseudorabies virus infection is provided.

2. The use according to claim 1, wherein the hippophae rhamnoides polysaccharide is capable of reducing cytopathic effects caused by porcine pseudorabies virus; and/or

3. The use according to claim 1, wherein the hippophae rhamnoides polysaccharide is capable of inhibiting porcine pseudorabies virus adsorption; and/or

4. The use of claim 1, wherein the porcine pseudorabies virus further comprises a variant porcine pseudorabies virus.

5. The use according to claim 4, wherein the strain of the variant porcine pseudorabies virus is PRV XJ5 strain.

6. Use according to claim 1, wherein the formulation comprises a pharmaceutical formulation, a disinfectant or a feed additive.

7. The use according to claim 6, wherein the pharmaceutical formulation further comprises a pharmaceutically acceptable salt and/or an adjuvant.

8. The use of claim 1, wherein the concentration of hippophae rhamnoides polysaccharide is 250 μ g/mL to 1000 μ g/mL.

9. The use according to claim 1, wherein the formulation is prepared by dissolving the hippophae rhamnoides polysaccharide in a solvent.

10. Use according to claim 9, wherein the solvent is PBS buffer.