Application of Anemarrhena asphodeloides polysaccharide in the preparation of preparations for preventing porcine epidemic diarrhea virus infection

Abstract

The present invention discloses the application of Anemarrhena polysaccharide in the preparation of an anti-porcine epidemic diarrhea virus infection preparation, which belongs to the field of antiviral application. The Anemarrhena polysaccharide exerts an anti-porcine epidemic diarrhea virus infection effect by inhibiting the adsorption and entry of porcine epidemic diarrhea virus into body cells. The present invention shows through in vitro cell experiments that Anemarrhena polysaccharide can effectively inhibit porcine epidemic diarrhea virus infection. By adding Anemarrhena polysaccharide at different stages of viral infection, it is found that Anemarrhena polysaccharide mainly inhibits the adsorption and entry of viruses into cells; the advantage of the Anemarrhena polysaccharide used in the present invention is that it is a traditional Chinese medicine extract, has little toxicity and side effects, and is highly safe. It can be used as a biological preparation for inhibiting porcine epidemic diarrhea virus infection, or as a feed additive, effectively preventing the continued prevalence of epidemic diarrhea in pig farms and reducing the economic losses of livestock breeding diseases.

Description

Technical Field

[0001] The invention relates to the field of antiviral applications, and in particular to the application of Anemarrhena asphodeloides polysaccharide in the preparation of a preparation for resisting porcine epidemic diarrhea virus infection. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious disease of pigs. It is an acute intestinal infectious disease of piglets and fattening pigs caused by Porcine Epidemic Diarrhea Virs (PEDV) and characterized by diarrhea, vomiting and dehydration in pigs of all ages. This disease is very similar to infectious gastroenteritis. Its main transmission route is feces-oral, but airborne transmission through the feces-nasal route may play a role in pig-to-pig and farm-to-farm transmission.

[0003] Porcine epidemic diarrhea virus belongs to the Coronavirus family and the Coronavirus genus. It is a single-stranded positive-strand RNA virus. The virus particles are polymorphic and tend to be spherical. They have an outer envelope with petal-like filaments on them. The inner part is a nucleoprotein core composed of RNA and nucleocapsid protein (N), which is a spiral structure with a diameter of 9 to 16 nm. Its genome length is 28,000 nucleotides (nt). Its main source of infection is sick pigs and the disease only occurs in pigs. Pigs of all ages can be infected and fall ill. The incidence rate of suckling pigs, piglets or fattening pigs is very high, especially suckling pigs. The main infection route of the disease is the cold season. Its main clinical symptoms are watery diarrhea or vomiting between diarrhea. Vomiting often occurs after eating or feeding. The severity of symptoms varies with age. The younger the age, the more severe the symptoms. Newborn piglets within one week of age will die of severe dehydration 3-4 days after diarrhea, with a mortality rate of up to 50%, and the highest mortality rate of 100%. The body temperature of sick pigs is normal or slightly high, they are depressed, and their appetite is reduced or lost. Weaned pigs and sows often show mental depression, anorexia and persistent diarrhea for about a week, and then gradually return to normal.

[0004] Since the early 1980s, porcine epidemic diarrhea has continued to be prevalent in my country's immune pig farms, which has brought great resistance to the purification of porcine epidemic diarrhea. Therefore, in order to reduce the economic losses caused by porcine epidemic diarrhea, it is urgent to develop new drugs to inhibit porcine epidemic diarrhea, which is not only conducive to solving current clinical problems, but also a new treatment option.

[0005] Many Chinese medicines have been proven to have immunomodulatory effects, and the traditional Chinese medicine Anemarrhena asphodeloides is one of them. Anemarrhena asphodeloides is bitter and cold in nature, and has the effects of clearing away heat and purging fire, nourishing yin and moistening dryness, quenching thirst and relieving restlessness. Pharmacological studies on Anemarrhena asphodeloides have shown that Anemarrhena asphodeloides has anti-platelet thrombosis, improves Alzheimer's disease, anti-tumor, anti-inflammatory, and antipyretic effects. Previous studies have isolated and purified the macromolecular component, Anemarrhena asphodeloides polysaccharide (TPA), from the rhizome of Anemarrhena asphodeloides. It is reported that Anemarrhena asphodeloides polysaccharide has antioxidant, bactericidal, anti-inflammatory, and anti-tumor effects, but the antiviral activity of Anemarrhena asphodeloides polysaccharide has not been reported. Summary of the invention

[0006] The purpose of the present invention is to provide an application of Anemarrhena asphodeloides polysaccharide in the preparation of an anti-porcine epidemic diarrhea virus infection preparation to solve the problems existing in the above-mentioned prior art. The present invention finds that Anemarrhena asphodeloides polysaccharide can inhibit the adsorption and entry of porcine epidemic diarrhea virus into cells, effectively inhibit porcine epidemic diarrhea virus infection, and Anemarrhena asphodeloides polysaccharide can be used as an antibacterial drug for the virus, providing a new idea for preventing the continued prevalence of epidemic diarrhea in immune pig farms and reducing the economic losses of livestock breeding diseases.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides application of Anemarrhena asphodeloides polysaccharide in preparing a preparation for resisting porcine epidemic diarrhea virus infection.

[0009] Furthermore, the Anemarrhena asphodeloides polysaccharide exerts an effect of resisting porcine epidemic diarrhea virus infection by inhibiting porcine epidemic diarrhea virus adsorption to cells.

[0010] Furthermore, the Anemarrhena asphodeloides polysaccharide exerts an anti-porcine epidemic diarrhea virus infection effect by inhibiting the porcine epidemic diarrhea virus from entering cells.

[0011] Furthermore, the Anemarrhena asphodeloides polysaccharide exerts an effect of resisting porcine epidemic diarrhea virus infection by inhibiting porcine epidemic diarrhea virus adsorption and entry into cells.

[0012] Furthermore, the preparation includes a biological preparation and an additive.

[0013] The present invention also provides the use of Anemarrhena asphodeloides polysaccharide in the preparation of porcine epidemic diarrhea virus adsorption cell inhibitor.

[0014] The present invention also provides the use of Anemarrhena asphodeloides polysaccharide in preparing an inhibitor of porcine epidemic diarrhea virus entry into cells.

[0015] The present invention discloses the following technical effects:

[0016] The present invention shows through in vitro cell experiments that Anemarrhena polysaccharide can effectively inhibit porcine epidemic diarrhea virus infection. The present invention adds Anemarrhena polysaccharide at different stages of virus infection and finds that Anemarrhena polysaccharide mainly inhibits virus adsorption and cell entry.

[0017] The advantage of the Anemarrhena asphodeloides polysaccharide used in the present invention is that it is a traditional Chinese medicine extract with little toxicity and side effects and high safety. It can be used as a biological agent for inhibiting porcine epidemic diarrhea virus, or as a feed additive to effectively prevent the continued prevalence of epidemic diarrhea in pig farms and reduce the economic losses caused by breeding diseases in animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The effect of Anemarrhena polysaccharide on porcine epidemic diarrhea virus infection in Vero cells; A is the effect of Anemarrhena polysaccharide on porcine epidemic diarrhea virus infection in Vero cells determined by Western blot; B is TCID 50 The effect of Anemarrhena polysaccharide on porcine epidemic diarrhea virus infection in Vero cells was measured; C was the effect of Anemarrhena polysaccharide on porcine epidemic diarrhea virus infection in Vero cells by indirect immunofluorescence assay;

[0020] Figure 2 The effect of Anemarrhena polysaccharide on the adsorption and cytosolic entry of porcine epidemic diarrhea virus on Vero cells, where A is the effect of Anemarrhena polysaccharide on the adsorption and cytosolic entry of porcine epidemic diarrhea virus on Vero cells determined by Western blot; B is TCID 50 The effect of Anemarrhena polysaccharide on the adsorption and cytotoxicity of porcine epidemic diarrhea virus on Vero cells was measured; C is the effect of Anemarrhena polysaccharide on the adsorption and cytotoxicity of porcine epidemic diarrhea virus on Vero cells by indirect immunofluorescence assay;

[0021] Figure 3 The effect of Anemarrhena polysaccharide on the adsorption of porcine epidemic diarrhea virus on Vero cells; A is the effect of Anemarrhena polysaccharide on the adsorption of porcine epidemic diarrhea virus on Vero cells determined by Western blot; B is TCID 50 The effect of Anemarrhena polysaccharide on the adsorption of porcine epidemic diarrhea virus on Vero cells was measured; C was the effect of Anemarrhena polysaccharide on the adsorption of porcine epidemic diarrhea virus on Vero cells by indirect immunofluorescence assay;

[0022] Figure 4 The effect of Anemarrhena polysaccharide on the entry of porcine epidemic diarrhea virus into Vero cells; A is the Western blot test of the effect of Anemarrhena polysaccharide on the entry of porcine epidemic diarrhea virus into Vero cells; B is TCID 50 The effect of Anemarrhena polysaccharide on the entry of porcine epidemic diarrhea virus into Vero cells was measured; C is the indirect immunofluorescence measurement of the effect of Anemarrhena polysaccharide on the entry of porcine epidemic diarrhea virus into Vero cells;

[0023] Figure 5 Western blot was used to determine the direct killing effect of Anemarrhena asphodeloides polysaccharide on porcine epidemic diarrhea virus;

[0024] Figure 6 Western blot was used to determine the effect of Anemarrhena asphodeloides polysaccharide pretreatment on Porcine Epidemic Diarrhea Virus infection in Vero cells. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The test materials used in the present invention: porcine epidemic diarrhea virus HLJBY strain, Vero cells (African green monkey kidney cells) are all stored in this laboratory. The present invention has no special requirements on the source of the Anemarrhena polysaccharide, and conventional commercial products in the field or self-preparation can be used. The Anemarrhena polysaccharide used in the following examples is specifically: Anemarrhena polysaccharide with a purity of 95% purchased from Yangling Ciyuan Biotechnology Co., Ltd. Weigh 0.05g of fine powder of Anemarrhena polysaccharide purchased from Yangling Ciyuan Biotechnology Co., Ltd. and dissolve it in 1mL PBS solution to obtain an anti-porcine epidemic diarrhea virus infection preparation.

[0031] The present invention shows through in vitro cell experiments that Anemarrhena polysaccharide can effectively inhibit porcine epidemic diarrhea virus infection. By adding Anemarrhena polysaccharide at different stages of virus infection (virus infection at 4°C is the virus adsorption stage, and the early stage of virus infection at 37°C is the virus entry stage), it is found that Anemarrhena polysaccharide mainly inhibits the adsorption and entry of viruses to cells. The specific research is as follows:

[0032] Example 1 Activity of Anemarrhena asphodeloides polysaccharide in inhibiting porcine epidemic diarrhea virus infection on Vero cells

[0033] (1) Western blot assay of the inhibitory activity of Anemarrhena asphodeloides polysaccharide on Vero cells against porcine epidemic diarrhea virus infection

[0034] Vero cells were digested with 0.25% trypsin and diluted with DMEM nutrient solution containing 5% fetal bovine serum and added dropwise to a 6-well plate at a concentration of 5 × 10 5 / well, and cultured in a 37°C, 5% CO2 incubator until the cells attached to the wall and formed a monolayer (about 17-18 hours), then discarded the original culture medium, washed the cells three times with PBS solution, and after absorbing the residual liquid, added 1 mL of serum-free DMEM and corresponding concentrations of Anemarrhena polysaccharides (0, 100 μg / mL, 200 μg / mL, 500 μg / mL) to each well, and incubated with Vero cells in a 37°C, 5% CO2 incubator for 1 hour, and then infected the cells with porcine epidemic diarrhea virus (0.1 MOI) and corresponding concentrations of Anemarrhena polysaccharides (0, 100 μg / mL, 200 μg / mL, 500 μg / mL). L, 200μg / mL, 500μg / mL) (Mock group is Vero cells without virus infection and without adding Anemarrhena polysaccharide, which is the negative control group), incubated in 37℃, 5% CO2 incubator for 1h, replaced with 2mL DMEM nutrient solution containing 2% fetal bovine serum per well and maintained with corresponding concentrations of Anemarrhena polysaccharide (0, 100μg / mL, 200μg / mL, 500μg / mL), incubated in 37℃, 5% CO2 incubator, 24h after infection, collect 1mL cell supernatant from each well (stored at -70℃ for later TCID 50 Experimental preparation), washed three times with PBS and then the residual liquid was sucked out, 2× protein loading was added to collect the cell sample, boiled in a metal bath at 96℃ for 15min, and then Western blot was performed. It was found that Anemarrhena polysaccharide reduced the expression of porcine epidemic diarrhea virus N protein ( Figure 1 Middle A), it was preliminarily confirmed that Anemarrhena asphodeloides polysaccharide inhibits the infection of porcine epidemic diarrhea virus.

[0035] (2)TCID 50 Determination of the inhibitory activity of Anemarrhena asphodeloides polysaccharide on Vero cells against porcine epidemic diarrhea virus infection

[0036] Vero cells were digested with 0.25% trypsin and diluted with DMEM nutrient solution containing 8% fetal bovine serum and added dropwise to a 96-well plate at a concentration of 2 × 10 3 / well is appropriate, and the cells are cultured in a 37°C, 5% CO2 incubator until the cells adhere to the wall and form a monolayer. The original culture medium is discarded, and the cells are washed three times with PBS. After the residual liquid is sucked out, the virus solution is diluted with serum-free DMEM to different concentrations and added to a 96-well plate containing Vero cells. Eight replicates are made for each concentration. After infection for 1.5 hours in a 37°C, 5% CO2 incubator, the plate is replaced with DMEM containing 2% fetal bovine serum for maintenance. After 72 hours of infection, the cell pathological changes are observed, and the TCID is calculated using the Reed-Muench method. 50 The results showed that Anemarrhena polysaccharide reduced the virus titer of porcine epidemic diarrhea virus infection supernatant ( Figure 1 Middle B) confirmed that Anemarrhena asphodeloides polysaccharide inhibits the infection of porcine epidemic diarrhea virus.

[0037] (3) IFA determination of the inhibitory activity of Anemarrhena polysaccharide on Vero cells against porcine epidemic diarrhea virus infection

[0038] Vero cells were digested with 0.25% trypsin and diluted with DMEM nutrient solution containing 5% fetal bovine serum and added dropwise to a 6-well plate at a concentration of 5 × 10 5 The concentration of each well is appropriate, and the cells are placed in a 37°C, 5% CO2 incubator and cultured until the cells adhere to the wall and form a monolayer (about 17-18 hours), then the original culture medium is discarded, and the cells are washed three times with PBS solution. After the residual liquid is sucked out, 1 mL of serum-free DMEM and corresponding concentrations of Anemarrhena polysaccharides (0, 100 μg / mL, 200 μg / mL, 500 μg / mL) are added to each well, and the Vero cells are placed in a 37°C, 5% CO2 incubator and incubated for 1 hour. The cells are infected with porcine epidemic diarrhea virus and have corresponding concentrations of Anemarrhena polysaccharides (0, 100 μg / mL, 200 μg / mL, 500μg / mL), incubated in a 37℃, 5% CO2 incubator for 1h, then replaced with 2mL of DMEM nutrient solution containing 2% fetal bovine serum per well and maintained with corresponding concentrations of Anemarrhena polysaccharide (0, 100μg / mL, 200μg / mL, 500μg / mL), incubated in a 37℃, 5% CO2 incubator. After infection for 24h, the cells were washed three times with PBS solution, fixed with 4% paraformaldehyde at 37℃ for 10min, then incubated with 0.1% TritonX-100 at 37℃ for 10 minutes, and then blocked with 5% BSA at 4℃ overnight. After blocking, PEDVN antibody was added and incubated at 37℃ for 1h, and then incubated with FITC-labeled goat anti-mouse IgG antibody (1:200) at 37℃ for 30 minutes, and observed after DAPI staining for 7 minutes. It was found that Anemarrhena polysaccharide had the activity of inhibiting porcine epidemic diarrhea virus infection ( Figure 1 Middle C).

[0039] Example 2 Effect of Anemarrhena asphodeloides polysaccharide on adsorption and cell entry of porcine epidemic diarrhea virus

[0040] (1) Western blot analysis of the effect of Anemarrhena asphodeloides polysaccharide on the adsorption and cellular entry of porcine epidemic diarrhea virus in Vero cells

[0041] Vero cells were digested with 0.25% trypsin and diluted to an appropriate density with DMEM nutrient solution containing 5% fetal bovine serum and added dropwise to a 6-well plate at 5×10 5 / well, cultured in a 37°C, 5% CO2 incubator until the cells adhered to the wall and formed a monolayer (about 17-18 hours), discarded the original culture medium, washed the cells three times with PBS solution, and after absorbing the residual liquid, added 1 mL of serum-free DMEM and corresponding concentrations of Anemarrhena polysaccharides (0, 100 μg / mL, 200 μg / mL, 500 μg / mL) to each well, and incubated with Vero cells in a 37°C, 5% CO2 incubator for 1 hour, discarded the liquid, replaced with 1 mL of cold serum-free DMEM in each well, infected with porcine epidemic diarrhea virus at 4°C and containing corresponding concentrations of Anemarrhena polysaccharides (0, 100 μg / mL, 200 μg / mL, 500 μg / mL) mL), after 1 hour, discard the liquid, wash three times with cold PBS to wash away the unabsorbed virus; then add 1mL of DMEM containing 2% fetal bovine serum and corresponding concentrations of Anemarrhena polysaccharide (0, 100μg / mL, 200μg / mL, 500μg / mL) to each well, incubate in a 37℃, 5% CO2 incubator for 1 hour, first wash three times with citric acid to wash away the virus that has not entered the cell, then wash three times with PBS and absorb the residual liquid, add 2mL of DMEM nutrient solution containing 2% fetal bovine serum to each well for maintenance, place in a 37℃, 5% CO2 incubator, and culture for 24 hours after infection, collect 1mL of cell supernatant from each well (stored at -70℃ for later TCID 50 Experimental preparation), add 2× protein loading to collect cell samples, boil in a metal bath at 96℃ for 15min, and then perform Western blot detection. The results are as follows Figure 2 As shown in A, it was found that Anemarrhena polysaccharide reduced the expression of porcine epidemic diarrhea virus N protein, and it was preliminarily confirmed that Anemarrhena polysaccharide inhibited the adsorption and entry of porcine epidemic diarrhea virus.

[0042] (2)TCID 50 Determination of the effect of Anemarrhena polysaccharide on the adsorption and entry of porcine epidemic diarrhea virus into Vero cells

[0043] Vero cells were digested with 0.25% trypsin and diluted with DMEM nutrient solution containing 8% fetal bovine serum and added dropwise to a 96-well plate at a concentration of 2 × 10 3 The concentration of each well is appropriate, and the cells are placed in a 37°C, 5% CO2 incubator to form a monolayer after the cells adhere to the wall. The original culture medium is discarded, and the cells are washed three times with PBS. After the residual liquid is sucked out, the virus solution is diluted with serum-free DMEM to different concentrations and added to a 96-well plate containing Vero cells. Eight replicates are made for each concentration, and the virus solution is placed in a 37°C, 5% CO2 incubator for 1.5 hours, and then replaced with DMEM containing 2% fetal bovine serum for maintenance. The cell pathological changes are observed after 72 hours of infection. The results are as follows Figure 2 As shown in B, it was found that Anemarrhena polysaccharide reduced the viral titer of porcine epidemic diarrhea virus infection, confirming that Anemarrhena polysaccharide inhibits the adsorption and cellular entry of porcine epidemic diarrhea virus.

[0044] (3) IFA determination of the effect of Anemarrhena polysaccharide on the adsorption and entry of porcine epidemic diarrhea virus into Vero cells

[0045] Vero cells were digested with 0.25% trypsin and diluted to an appropriate density with DMEM nutrient solution containing 5% fetal bovine serum and added dropwise to a 6-well plate at 5×10 5 / well is appropriate, and the cells are placed in a 37°C, 5% CO2 incubator and cultured until the cells adhere to the wall and form a monolayer (about 17-18h), the original culture medium is discarded, the cells are washed three times with PBS solution, and after the residual liquid is sucked out, 1mL of serum-free DMEM and corresponding concentrations of Anemarrhena polysaccharides (100μg / mL, 200μg / mL, 500μg / mL) are added to each well, and the Vero cells are placed in a 37°C, 5% CO2 incubator and incubated for 1h, the liquid is discarded, and each well is replaced with 1mL of cold serum-free DMEM, and porcine epidemic diarrhea virus is infected at 4°C and corresponding concentrations of Anemarrhena polysaccharides (100μg / mL, 200μg / mL, 500μg / mL) are present. After incubation for 1h, the liquid is discarded, and the cells are washed three times with cold PBS to wash away Unadsorbed virus; then 1 mL of DMEM containing 2% fetal bovine serum and corresponding concentrations of Anemarrhena asphodeloides polysaccharide (100 μg / mL, 200 μg / mL, 500 μg / mL) was added to each well, and incubated in a 37°C 5% CO2 incubator for 1 h, first washed three times with citric acid to wash away the virus that did not enter the cell, then washed three times with PBS and the residual liquid was absorbed, and 2 mL of DMEM nutrient solution containing 2% fetal bovine serum was added to each well for maintenance, and cultured in a 37°C, 5% CO2 incubator. After 24 h of infection, the supernatant was collected, the cells were washed three times with PBS solution, fixed with 4% paraformaldehyde at 37°C for 10 min, and then incubated with 0.1% TritonX-100 at 37°C for 10 minutes, and then blocked with 5% BSA at 4°C overnight. After blocking, PEDVN antibody was added and incubated at 37°C for 1 hour, and then incubated with FITC-labeled goat anti-mouse IgG antibody (1:200) at 37°C for 30 minutes, and observed after DAPI staining for 7 minutes. Figure 2 Middle C).

[0046] Example 3 Effect of Anemarrhena asphodeloides polysaccharide on adsorption of porcine epidemic diarrhea virus

[0047] (1) Western blot analysis of the effect of Anemarrhena asphodeloides polysaccharide on the adsorption of porcine epidemic diarrhea virus on Vero cells

[0048] Vero cells were digested with 0.25% trypsin and diluted with DMEM nutrient solution containing 5% fetal bovine serum and added dropwise to a 6-well plate at a concentration of 5 × 10 5The concentration of each well is appropriate, and the cells are placed in a 37°C, 5% CO2 incubator and cultured until the cells adhere to the wall and form a monolayer (about 17-18 hours), then the original culture medium is discarded, and the cells are washed three times with PBS solution. After the residual liquid is sucked out, 1 mL of serum-free DMEM is added to each well to dilute and add the corresponding concentration of Anemarrhena asphodeloides polysaccharide (100 μg / mL, 200 μg / mL, 500 μg / mL), and the cells are placed in a 37°C, 5% CO2 incubator for 1 hour, and then washed three times with cold PBS, and 1 mL of serum-free DMEM is added to each well. Clear cold DMEM, add porcine epidemic diarrhea virus and corresponding concentrations of Anemarrhena asphodeloides polysaccharide (100μg / mL, 200μg / mL, 500μg / mL), incubate at 4℃ for 1h, discard the liquid, wash three times with cold PBS to wash away the unadsorbed virus; then replace with DMEM nutrient solution containing 2mL2% fetal bovine serum per well for maintenance, culture in a 37℃, 5% CO2 incubator, and after 24h of infection, collect 1mL of cell supernatant from each well (store at -70℃ for later TCID 50 The cells were washed three times with PBS, the residual liquid was sucked out, 2× protein loading was added to collect the cell samples, boiled in a metal bath at 96°C for 15 min, and then tested by Western blot. Figure 3 As shown in A, it was found that Anemarrhena polysaccharide reduced the expression of porcine epidemic diarrhea virus N protein, confirming that Anemarrhena polysaccharide inhibits the adsorption of porcine epidemic diarrhea virus.

[0049] (2)TCID 50 Determination of the effect of Anemarrhena polysaccharide on the adsorption of porcine epidemic diarrhea virus on Vero cells

[0050] Vero cells were digested with 0.25% trypsin and diluted with DMEM nutrient solution containing 8% fetal bovine serum and added dropwise to a 96-well plate at a concentration of 2 × 10 3 The concentration of each well is appropriate, and the cells are placed in a 37°C, 5% CO2 incubator to form a monolayer after the cells adhere to the wall. The original culture medium is discarded, and the cells are washed three times with PBS. After the residual liquid is sucked out, the virus solution is diluted with serum-free DMEM to different concentrations and added to a 96-well plate containing Vero cells. Eight replicates are made for each concentration, and the virus solution is placed in a 37°C, 5% CO2 incubator for 1.5 hours, and then replaced with DMEM containing 2% fetal bovine serum for maintenance. The cell pathological changes are observed after 72 hours of infection. The results are as follows Figure 3 As shown in B, it was found that Anemarrhena polysaccharide reduced the virus titer of porcine epidemic diarrhea virus infection, confirming that Anemarrhena polysaccharide inhibits the adsorption of porcine epidemic diarrhea virus.

[0051] (3) IFA determination of the effect of Anemarrhena polysaccharide on the adsorption of porcine epidemic diarrhea virus on Vero cells

[0052] Vero cells were digested with 0.25% trypsin and diluted with DMEM nutrient solution containing 5% fetal bovine serum and added dropwise to a 6-well plate at a concentration of 5 × 10 5 The concentration of each well is appropriate, and the cells are cultured in a 37°C, 5% CO2 incubator until the cells adhere to the wall and form a monolayer (about 17-18 hours), then the original culture medium is discarded, and the cells are washed three times with PBS solution. After the residual liquid is sucked out, 1 mL of serum-free DMEM is added to each well to dilute and add the corresponding concentration of Anemarrhena asphodeloides polysaccharides (100 μg / mL, 200 μg / mL, 500 μg / mL), and the cells are placed in a 37°C, 5% CO2 incubator for 1 hour, and then washed three times with cold PBS, 1 mL of cold DMEM without fetal bovine serum is added to each well, and porcine epidemic diarrhea virus and the corresponding concentration of Anemarrhena asphodeloides polysaccharides are added. In the presence of Anemarrhena polysaccharide (100μg / mL, 200μg / mL, 500μg / mL), incubate at 4℃ for 1h, discard the liquid, wash three times with cold PBS to wash away the unadsorbed virus; replace with DMEM nutrient solution containing 2mL2% fetal bovine serum in each well for maintenance, and culture in a 37℃, 5% CO2 incubator. After 24h of infection, collect the supernatant, wash the cells three times with PBS solution, add 4% paraformaldehyde and fix at 37℃ for 10min, then incubate with 0.1% TritonX-100 at 37℃ for 10 minutes, and then add 5% BSA to block at 4℃ overnight. After blocking, add PEDVN antibody and incubate at 37℃ for 1h, then incubate with FITC-labeled goat anti-mouse IgG antibody (1:200) at 37℃ for 30 minutes, and observe after DAPI staining for 7 minutes. It was found that Anemarrhena polysaccharide can inhibit the adsorption of porcine epidemic diarrhea virus ( Figure 3 Middle C).

[0053] Example 4 Effect of Anemarrhena asphodeloides polysaccharide on porcine epidemic diarrhea virus entry into cells

[0054] (1) Western blot analysis of the effect of Anemarrhena asphodeloides polysaccharide on the entry of porcine epidemic diarrhea virus into Vero cells

[0055] Vero cells were digested with 0.25% trypsin and diluted to an appropriate density with DMEM nutrient solution containing 5% fetal bovine serum and added dropwise to a 6-well plate at a density of 5 × 10 5The concentration of each well is appropriate, and the cells are placed in a 37°C, 5% CO2 incubator and cultured until the cells adhere to the wall and form a monolayer (about 17-18 hours), then the original culture medium is discarded, and the cells are washed three times with cold PBS solution, and the residual liquid is sucked out. 1mL of serum-free cold DMEM is added to each well and infected with porcine epidemic diarrhea virus. After incubation at 4°C for 1 hour, the original culture medium is discarded, and the cells are washed three times with cold PBS solution and the residual liquid is sucked out. 1mL of cold DMEM containing 2% fetal bovine serum is added to each well and the corresponding The cells were incubated at 37°C for 1 h in the presence of 100 μg / mL, 200 μg / mL, and 500 μg / mL of Anemarrhena asphodeloides polysaccharide. The cells were washed three times with citric acid to remove the virus that did not enter the cells, and then washed three times with PBS and the residual liquid was aspirated. Each well was replaced with 2 mL of DMEM nutrient solution containing 2% fetal bovine serum and maintained in a 37°C, 5% CO2 incubator. After 24 h of infection, 1 mL of cell supernatant was collected from each well (stored at -70°C for later TCID 50 Experimental preparation), add 2× protein loading to collect cell samples, boil in a metal bath at 96°C for 15 min, and then perform Western blot detection. The results are as follows Figure 4 As shown in A, it was found that Anemarrhena polysaccharide reduced the expression of porcine epidemic diarrhea virus N protein, confirming that Anemarrhena polysaccharide inhibits the entry of porcine epidemic diarrhea virus into the cells.

[0056] (2)TCID 50 Determination of the effect of Anemarrhena polysaccharide on the entry of porcine epidemic diarrhea virus into Vero cells

[0057] Vero cells were digested with 0.25% trypsin and diluted with DMEM nutrient solution containing 8% fetal bovine serum and added dropwise to a 96-well plate at a concentration of 2×10 3 / well, culture in a 37℃, 5% CO2 incubator until the cells adhere to the wall and form a monolayer, discard the original culture medium, wash three times with PBS, absorb the residual liquid, dilute the virus solution with serum-free DMEM to different concentrations, add it to a 96-well plate containing Vero cells, do 8 replicates for each concentration, place it in a 37℃, 5% CO2 incubator for 1.5h, and then change it to DMEM containing 2% fetal bovine serum for maintenance, and observe the cell pathological changes after 72h of infection. The results are as follows Figure 4 As shown in B, it was found that Anemarrhena polysaccharide reduced the virus titer of porcine epidemic diarrhea virus infection, confirming that Anemarrhena polysaccharide inhibits the entry of porcine epidemic diarrhea virus into the cells.

[0058] (3) IFA determination of the effect of Anemarrhena polysaccharide on the entry of porcine epidemic diarrhea virus into Vero cells

[0059] Vero cells were digested with 0.25% trypsin and diluted to an appropriate density with DMEM nutrient solution containing 5% fetal bovine serum and added dropwise to a 6-well plate at a density of 5 × 10 5The concentration of each well is appropriate, and the cells are cultured in a 37°C, 5% CO2 incubator until the cells adhere to the wall and form a monolayer (about 17-18 hours), then the original culture medium is discarded, and the cells are washed three times with cold PBS solution, and the residual liquid is sucked out. 1mL of serum-free cold DMEM is added to each well and infected with porcine epidemic diarrhea virus. After incubation at 4°C for 1 hour, the original culture medium is discarded, and the cells are washed three times with cold PBS solution and the residual liquid is sucked out. 1mL of cold DMEM containing 2% fetal bovine serum and corresponding concentrations of Anemarrhena asphodeloides polysaccharide (100μg / mL, 200μg / mL, 5 00μg / mL), incubated at 37℃ for 1h, the cells were first washed three times with citric acid to wash away the virus that did not enter the cells, then washed three times with PBS and the residual liquid was aspirated, and each well was replaced with 2mL of DMEM nutrient solution containing 2% fetal bovine serum for maintenance, and cultured in a 37℃, 5% CO2 incubator. After infection for 24h, the supernatant was collected, the cells were washed three times with PBS solution, 4% paraformaldehyde was added and fixed at 37℃ for 10min, and then incubated with 0.1% TritonX-100 at 37℃ for 10 minutes, and then 5% BSA was added and blocked at 4℃ overnight. After blocking, PEDVN antibody was added and incubated at 37℃ for 1h, and then incubated with FITC-labeled goat anti-mouse IgG antibody (1:200) at 37℃ for 30 minutes, and observed after DAPI staining for 7 minutes. It was found that Anemarrhena asphodeloides polysaccharide inhibited the entry of porcine epidemic diarrhea virus into the cells ( Figure 4 Middle C).

[0060] Example 5 Direct killing effect of Anemarrhena asphodeloides polysaccharide on porcine epidemic diarrhea virus

[0061] Vero cells were digested with 0.25% trypsin and diluted with DMEM nutrient solution containing 5% fetal bovine serum and added dropwise to a 6-well plate at a concentration of 5 × 10 5 The concentration of each well is appropriate, and the cells are placed in a 37°C, 5% CO2 incubator and cultured until the cells adhere to the wall and form a monolayer (about 17-18 hours). The original culture medium is discarded, and the cells are washed three times with PBS solution. After the residual liquid is sucked out, PBS or Anemarrhena polysaccharide solution (100μg / mL, 200μg / mL, 500μg / mL) is incubated with porcine epidemic diarrhea virus at 37°C for 1 hour, and the liquid is discarded. The cells are washed three times with PBS and the residual liquid is sucked out. 2mL of DMEM containing 2% fetal bovine serum is added to each well and placed in a 37°C, 5% CO2 incubator. 2× protein loading is added 24 hours after infection to collect cell samples, boiled in a metal bath at 96°C for 15 minutes, and then Western blot detection is performed. The results are as follows Figure 5 As shown, it was found that Anemarrhena polysaccharide could not directly kill porcine epidemic diarrhea virus. Combined with Examples 1-4, it can be seen that by adding Anemarrhena polysaccharide at different stages of virus infection, it was found that Anemarrhena polysaccharide mainly inhibited the adsorption and entry of viruses into cells, effectively inhibited virus infection, and can be used as an antiviral biological preparation or feed additive.

[0062] Example 6 Effect of pretreatment of Vero cells with Anemarrhena asphodeloides polysaccharide on porcine epidemic diarrhea virus infection.

[0063] Vero cells were digested with 0.25% trypsin and diluted with DMEM nutrient solution containing 5% fetal bovine serum and added dropwise to a 6-well plate at a concentration of 5 × 10 5 The concentration of each well is appropriate, and the cells are placed in a 37°C, 5% CO2 incubator and cultured until the cells adhere to the wall and form a monolayer (about 17-18 hours). The original culture medium is discarded, and the cells are washed three times with PBS solution. After the residual liquid is sucked out, 1 mL of serum-free DMEM and the corresponding concentration of Anemarrhena polysaccharide (100 μg / mL, 200 μg / mL, 500 μg / mL) are added to each well. After incubation in a 37°C, 5% CO2 incubator for 1 hour, the liquid is discarded, and the cells are washed three times with PBS solution and the residual liquid is sucked out 1 mL of serum-free DMEM was added to each well and infected with porcine epidemic diarrhea virus. After incubation at 37°C and 5% CO2 for 1 hour, the liquid was discarded, the cells were washed three times with PBS and the residual liquid was sucked out. 2 mL of DMEM containing 2% fetal bovine serum was added to each well and incubated at 37°C and 5% CO2. After 24 hours of infection, 2× protein loading was added to collect cell samples, boiled in a metal bath at 96°C for 15 minutes, and then Western blot was performed. The results are as follows Figure 6 As shown, polysaccharide pretreatment of Vero cells has no effect on subsequent virus infection. Combined with the content of the above examples, it is clear that only when Anemarrhena asphodeloides polysaccharide is added at different stages of virus infection can it effectively inhibit porcine epidemic diarrhea virus infection.

[0064] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of Anemarrhena asphodeloides polysaccharide as the sole active ingredient in the preparation of preparations against porcine epidemic diarrhea virus infection.

2. The use according to claim 1, characterized in that: The Anemarrhena asphodeloides polysaccharide inhibits porcine epidemic diarrhea virus infection by inhibiting porcine epidemic diarrhea virus adsorption to cells.

3. The use according to claim 1, characterized in that: The Anemarrhena asphodeloides polysaccharide inhibits porcine epidemic diarrhea virus infection by inhibiting porcine epidemic diarrhea virus from entering cells.

4. The use according to claim 1, characterized in that: The Anemarrhena asphodeloides polysaccharide inhibits porcine epidemic diarrhea virus infection by inhibiting the adsorption and entry of porcine epidemic diarrhea virus into cells.

5. The use according to claim 1, characterized in that: The preparation includes a biological preparation and an additive.

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