阅读说明：本技术 一种抗猪伪狂犬病病毒的药物、消毒剂、添加剂及其使用方法 (Medicine, disinfectant and additive for resisting porcine pseudorabies virus and using method thereof ) 是由 郇长超 姚婧婷 李晓丽 于亚玲 高崧 于 2021-06-28 设计创作，主要内容包括：本发明公开了一种抗猪伪狂犬病病毒的药物、消毒剂、添加剂及其使用方法,其中,药物、消毒剂,添加剂为槐耳多糖,本发明提供的槐耳多糖对于猪伪狂犬病病毒有着良好的抑制、预防的效果。(The invention discloses a medicament, a disinfectant, an additive and a using method thereof for resisting porcine pseudorabies virus, wherein the medicament, the disinfectant and the additive are pagodatree ear polysaccharide.)

1. A medicine for resisting porcine pseudorabies virus is characterized in that: the medicine is pagodatree ear polysaccharide.

2. An additive for resisting porcine pseudorabies virus is characterized in that: the medicine is pagodatree ear polysaccharide.

3. A disinfectant for resisting porcine pseudorabies virus is characterized in that: the disinfectant is pagodatree fungus polysaccharide.

4. The agent, disinfectant or additive for treating porcine pseudorabies virus according to claims 1, 2 or 3, wherein: the medicine, disinfectant and additive are pagodatree fungus polysaccharide.

5. The application method of the medicine, the disinfectant and the additive for resisting the porcine pseudorabies virus is characterized in that: the medicine, disinfectant and additive are used for resisting the porcine pseudorabies virus variant XJ 5.

6. The use method of the anti-porcine pseudorabies virus drugs, disinfectants and additives according to claim 5, characterized in that: the medicine, the disinfectant and the additive are used in the adsorption process of the porcine pseudorabies virus variant XJ 5.

7. The use method of the anti-porcine pseudorabies virus drugs, disinfectants and additives according to claim 5, characterized in that: the medicine, the disinfectant and the additive are used for the cell entering process of the porcine pseudorabies virus variant XJ 5.

8. The use method of the anti-porcine pseudorabies virus drugs, disinfectants and additives according to claim 5, characterized in that: the medicine, the disinfectant and the additive are used in the virus replication process of the porcine pseudorabies virus variant XJ 5.

9. The use method of the anti-porcine pseudorabies virus drugs, disinfectants and additives according to claim 5, characterized in that: the concentration of the medicines, disinfectors and additives is 25-200 mug/ml.

10. The use of the anti-porcine pseudorabies virus medicament, disinfectant, additive according to claim 5 or 9, characterized in that: the concentration of the medicines, disinfectants and additives is 200 mug/ml.

Technical Field

The invention relates to the technical field of biological preparations, in particular to a medicament, a disinfectant and an additive for resisting porcine pseudorabies virus and a using method thereof.

Background

Porcine Pseudorabies (PR) is a disease of pigs caused by infection of the herd with Pseudorabies virus, and was first transmitted to pigs by cattle. With the scale enlargement of intensive pig farms, the global development of international trade in animals and their by-products, and the increase in the demand for domestic live pig transportation flow, the disease has been an explosive epidemic. The disease is caused by oral and nasal infection, and further invades respiratory tract, tonsil, hyperpyrexia, intolerance of cold, mental depression, hoarseness, reduction of intake water volume, cerebral sulcus hemorrhage caused by central nerve damage, and ataxia during current step, pulmonary edema, tonsil swelling, liver, kidney and spleen surface are white and bad. The pregnant sow has abortion, dead fetus, mummy fetus, and high sterility and adverse condition rate after repeated mating. After 84-day-old framed pigs are infected, the weight gain of a pig group is mainly influenced except for typical clinical symptoms, and huge losses are caused to a farm. In China, the Bartha-K61 vaccine was introduced from Hungarian in 1987 for porcine pseudorabies virus, and in the following years, the domestic Bartha-K61 vaccine also appeared in succession. Before 2011, the Bartha-K61 vaccine used in China for resisting porcine pseudorabies can well control the occurrence of the disease in a swinery, but with the outbreak of a new pseudorabies epidemic situation of the swinery immunized by the vaccine after 2011, the investigation shows that due to the occurrence of various variant strains, the widely used Bartha-K61 vaccine has only 50-60% of protection power on the swinery, the death rate of 12-week-old pigs is close to 100%, and the virulence of the variant strains on the swinery is abnormally enhanced. The anti-variant strain appears, the median lethal dose of the Bartha-K61 vaccine virus content used in China is 5000, so the inoculation of the Bartha-K61 vaccine made in China cannot protect the swinery with high quality, the effect of clearing the wild strain and finally purifying the pseudorabies swinery can be achieved only by purchasing imported Bartha-K61 vaccine (the median lethal dose of the virus content is 105) immunization and carpet immunization of the swinery to achieve the aim of clearing the wild strain, although the African swine fever is the most urgent problem to be solved in the pig breeding industry in China, the generation of the pseudorabies can not be avoided, and once the pseudorabies occurs in the swinery, the direct and indirect economic loss is brought to the development of the pig breeding industry in the African swine fever background in China.

Porcine Pseudorabies virus (PRV) belongs to herpesviridae and herpesviridae A, virulence is cooperatively controlled by a plurality of genes, PRV host is wide, replication cycle is short, and pathogenicity is high. Structurally, the capsule is divided into 4 layers from inside to outside, namely double-stranded DNA, nucleocapsid, protein and cyst membrane. The UL segment of the PRV genome distributes multiple virulence genes, such as gB, gC, etc., which, together with other genes, determine the virulence of the virus. The gB gene has a full length of 2742bp, is a main virulence gene of PRV, can induce an organism to generate 2 neutralizing antibodies, and can ensure that PRV particles and host cells are fused to finish the process of invading the host cells. At present, in domestic markets, various vaccines which are developed by taking classical strains as parents, but because the virus has brand-new variation on the genome level, the traditional vaccine can not enable swinery to completely resist PR, and the development of a biological agent for preventing and/or treating the pseudorabies of the variant pigs which are popular at present is urgent and necessary. Avoids the possible hepatic first pass effect and gastrointestinal inactivation of oral administration, thereby avoiding many side effects.

The invention provides a medicament, a disinfectant and an additive for resisting porcine pseudorabies virus and application thereof, compared with other medicaments with antiviral effect, the medicament has the following advantages:

1. the pagodatree ear polysaccharide is a medicinal fungus, has a long history in clinical treatment, is mainly used for treating human cancers and inflammations, has a main active component of proteoglycan, and can inhibit the growth and metastasis of tumors through various ways.

2. The pagodatree ear polysaccharide has little pollution to environment after being metabolized by organism

3. The pagodatree ear polysaccharide has obvious effect of resisting the infection of the variant strain XJ5 porcine pseudorabies virus to cells, and tests show that the medicine can reduce cytopathic effect caused by the virus.

4. The concentration of the pagodatree ear polysaccharide is 25-200 mug/ml, and the infection of the porcine pseudorabies virus variant virus XJ5 can be prevented by combining with a receptor on the cell surface.

5. The concentration of the pagodatree ear polysaccharide is 25-200 mug/ml, and the effect of resisting virus infection is achieved by influencing the adsorption effect of the virus.

6. The concentration of the pagodatree ear polysaccharide is 25-50 mug/ml, so that the cell-entering effect of the virus is influenced.

7. The concentration of the pagodatree ear polysaccharide is 25-200 mug/ml, so that the replication effect of the virus is influenced.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a graph showing the effect of pagodatree ear polysaccharide on cytopathic effect of porcine pseudorabies virus variant XJ5 infected PK-15 cells under a microscope;

FIG. 2 shows the effect of trametes robiniophila polysaccharide on PK-15 cells on infection with a variant strain XJ5 of porcine pseudorabies virus;

wherein A is Western blot for determining the influence of pagodatree ear polysaccharide on PK-15 cells on infection of a porcine pseudorabies virus variant XJ 5; b is TCID50 to determine the influence of pagodatree ear polysaccharide on PK-15 cells on infection of porcine pseudorabies virus variant XJ 5; c is the influence of immunofluorescence antibody assay (IFA) pagodatree ear polysaccharide on PK-15 cells on infection of the porcine pseudorabies virus variant XJ 5;

FIG. 3 shows the effect of pagodatree ear polysaccharide on PK-15 cells on the adsorption and entry of porcine pseudorabies virus variant XJ5,

in the figure, A is a Western blot for determining the influence of pagodatree ear polysaccharide on PK-15 cells on the adsorption and cell entry of a porcine pseudorabies virus variant XJ 5; b is TCID50 for determining the influence of pagodatree ear polysaccharide on PK-15 cells on the adsorption and cell entry of the porcine pseudorabies virus variant XJ 5; c is the influence of immunofluorescence antibody assay (IFA) pagodatree ear polysaccharide on PK-15 cells on the adsorption and cell entry of the porcine pseudorabies virus variant XJ 5;

FIG. 4 shows the effect of pagodatree ear polysaccharide on the adsorption of porcine pseudorabies virus variant XJ5 on PK-15 cells;

in the figure, A is a Western blot for determining the influence of pagodatree ear polysaccharide on PK-15 cells on the adsorption of a porcine pseudorabies virus variant XJ 5; b is TCID50 to determine the influence of pagodatree ear polysaccharide on PK-15 cells to the adsorption of porcine pseudorabies virus variant XJ 5; c is the influence of immunofluorescence antibody assay (IFA) pagodatree ear polysaccharide on PK-15 cells on the adsorption of the porcine pseudorabies virus variant XJ 5;

FIG. 5 shows the effect of pagodatree ear polysaccharide on PK-15 cells on the invasion of porcine pseudorabies virus variant XJ 5;

in the figure, A is a Western blot for determining the influence of pagodatree ear polysaccharide on PK-15 cells on porcine pseudorabies virus encytosis; b is the result of the gray level analysis of the result of FIG. 5A; c is TCID50 for determining the influence of pagodatree ear polysaccharide on PK-15 cells on the entrance of a porcine pseudorabies virus variant XJ 5; d is the influence of the immune fluorescent antibody assay (IFA) pagodatree ear polysaccharide on the PK-15 cells on the cell entrance of the porcine pseudorabies virus variant XJ 5;

FIG. 6 is a Western blot for determining the influence of pagodatree ear polysaccharide on PK-15 cells on 4h replication of porcine pseudorabies virus variant XJ5 virus infection;

FIG. 7 is a Western blot assay for determining the influence of pagodatree ear polysaccharide on PK-15 cells on 6h replication of porcine pseudorabies virus variant XJ5 virus infection;

FIG. 8 shows that Western blot detects that pagodatree ear polysaccharide is possibly combined with cell surface receptors on PK-15 cells to play a role in preventing infection of porcine pseudorabies virus variant XJ5 strain.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1

PK-15 cells recovered from this laboratory were placed at 37 ℃ in the presence of 5% CO in DMEM containing 5% fetal bovine serum₂Cultured in an incubator and subcultured to the third generation at 5X 10 per well⁵Uniformly spreading the concentration of each cell in a 6-well plate, after culturing to a logarithmic growth phase for about 16h, when the cells grow to about 70-75% volume of each well, discarding DMEM containing 5% fetal calf serum, washing with PBS buffer solution for three times, completely sucking the rest PBS in the last time, adding empty DMEM 1ml, diluting the dissolved pagodatree ear polysaccharide to 25mg, 50mg,100mg and 200mg, incubating PRV XJ5(MOI is 0.1) and the cells for 1h, changing to DMEM solution containing 2% fetal calf serum 2ml after 1h, adding PBS buffer solution or pagodatree ear polysaccharide (25mg, 50mg,100mg and 200mg) with corresponding concentration, and observing the cell morphology and pathological changes under a microscope for 12h and 24h respectively. As shown by the results in fig. 1, pagodatree ear polysaccharide can reduce the cytopathic effect caused by PRV XJ 5. The pagodatree ear polysaccharide can reduce the infection of the variant porcine pseudorabies virus XJ 5.

Example 2

(1) Western blot detection of the activity of pagodatree ear polysaccharide in inhibiting infection of porcine pseudorabies virus variant strain XJ5 on PK-15 cells:

PK-15 cells were digested with trypsin and then diluted with a DMEM nutrient solution containing fetal bovine serum at a volume concentration of 5% to 5X 10⁵The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO₂After the cells adhered to form a monolayer in the incubator are positioned after the logarithmic growth phase (about 17h), the cells are washed for three times by PBS buffer solution, after residual PBS is absorbed out, pagodatree ear polysaccharide (25 mug, 50 mug, 100 mug and 200 mug) and porcine pseudorabies virus variant XJ5 strain which are diluted to corresponding concentrations by 1mL of serum-free DMEM are added and mixed with PK-15 cells at 37 ℃ and 5% CO₂After the incubator is incubated for 1h, 2mL of DMEM nutrient solution containing 2% fetal calf serum is replaced to maintain the pagodatree ear with corresponding concentrationPolysaccharide (25. mu.g, 50. mu.g, 100. mu.g, 200. mu.g) was present at 37 ℃ in the presence of 5% CO₂Culturing in an incubator, after infecting for 24h, collecting cell supernatant (1mL is stored at-70 ℃ for later TCID50 experiment preparation), washing with PBS buffer solution for three times, sucking up residual liquid, adding 2 x protein loading to collect cell samples, boiling for 15min at 96 ℃ in a metal bath, and carrying out Western blot detection. The sophorae ear polysaccharide is found to reduce the expression of porcine pseudorabies virus gB protein (A in figure 2), and the sophorae ear polysaccharide is preliminarily proved to reduce the infection of porcine pseudorabies virus variant XJ5 strain.

(2) TCID50 determination of the activity of pagodatree ear polysaccharide in inhibiting infection of porcine pseudorabies virus variant XJ5 on PK-15 cells:

after being digested by Vero cell pancreatin, the Vero cell is diluted by DMEM nutrient solution of fetal bovine serum with volume concentration of 8 percent and is diluted by 2 multiplied by 10³The concentration of each well was added dropwise to a 96-well plate and the plate was placed at 37 ℃ in 5% CO₂After the cells adhere to the wall to form a monolayer and are in a logarithmic growth phase (about 16 hours), the cells are washed for three times by PBS buffer solution, after residual liquid is completely sucked, virus liquid with different concentrations diluted by serum-free DMEM is added, each concentration is repeated for 4 times, after 1.5 hours of infection, the cells are maintained by changing into the DMEM containing 2% fetal calf serum, and the condition of cytopathic effect is observed after 72 hours of infection. It was found that the sophorae ear polysaccharide reduced the virus titer of the supernatant from infection with the porcine pseudorabies virus variant XJ5 strain (B in FIG. 2), confirming that the sophorae ear polysaccharide reduced the infection with the porcine pseudorabies virus variant XJ5 strain.

(3) Indelect immunology Assay (IFA) Assay of the activity of sophorae ear polysaccharide to inhibit infection of the porcine pseudorabies virus variant XJ5 strain on PK-15 cells:

PK-15 cells were digested with trypsin and then diluted with 10% fetal bovine serum by volume in DMEM medium at 5X 10⁵The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO₂After the cells adhered to form a monolayer in the incubator are positioned after the logarithmic growth phase (about 17h), the cells are washed for three times by PBS buffer solution, after residual PBS is absorbed out, pagodatree ear polysaccharide (25 mug, 50 mug, 100 mug and 200 mug) and porcine pseudorabies virus variant XJ5 strain which are diluted to corresponding concentrations by 1mL of serum-free DMEM are added and mixed with PK-15 cells at 37 ℃ and 5% CO₂After incubation in the incubator for 1h, 2mL of the medium containing 2%DMEM nutrient solution of fetal calf serum is maintained and corresponding concentration of trametes robiniophila polysaccharide (25 μ g, 50 μ g,100 μ g, 200 μ g) exists, and the mixture is placed at 37 deg.C and 5% CO₂Culturing in an incubator, after infecting for 24h, removing supernatant, washing for three times by using PBS buffer solution, completely sucking residual PBS for the last time, adding 4% paraformaldehyde capable of covering the cell surface and fixing for 15min at 37 ℃, then penetrating cells for 10 min by using 0.1% TritonX-100, then washing for three times by using the PBS buffer solution, completely sucking for the last time, adding 5% BSA blocking solution for 1.5h at 37 ℃ or overnight at 4 ℃, then washing for three times by using PBS, completely sucking for the last time, and mixing with PRV-resistant pig positive serum 1: 200 dilutions were incubated at 37 ℃ for 2h, washed three times with PBST, blotted out for the last time, and incubated with 1: 200 diluted secondary goat anti-pig IgG is incubated for 1h at 37 ℃, protected from light, dyed with DAPI for 5min, washed with PBST for three times, completely blotted in the last time, and placed under a fluorescence microscope to observe cell morphology and lesion (C in figure 2), thereby confirming that the pagoda ear polysaccharide reduces the infection of the porcine pseudorabies virus variant XJ5 strain.

Example 3

(1) Western blot detection shows that pagodatree ear polysaccharide inhibits the adsorption and encytosis of porcine pseudorabies virus variant strain XJ5 on PK-15 cells:

PK-15 cells were digested with trypsin and then diluted with a DMEM nutrient solution containing fetal bovine serum at a volume concentration of 5% to 5X 10⁵The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO₂After the cells are adhered to form a monolayer and are positioned after the logarithmic growth phase (about 17 hours) in the incubator, the cells are washed for three times by PBS buffer solution at 4 ℃, residual PBS is absorbed out, pagodatree ear polysaccharide (25 mu g, 50 mu g,100 mu g and 200 mu g) and porcine pseudorabies virus variant strain XJ5 which are diluted to corresponding concentrations by 1mL of serum-free DMEM at 4 ℃ are added, the cells are incubated for 1 hour with PK-15 cells at 4 ℃, then 2mL of DMEM nutrient solution containing 2% fetal calf serum is replaced and placed at 37 ℃ and 5% CO₂Incubating in incubator for 1 hr in the presence of corresponding concentration of trametes robiniophila polysaccharide (25 μ g, 50 μ g,100 μ g, 200 μ g), and placing at 37 deg.C and 5% CO₂Culturing in incubator, washing with citric acid for three times, washing with PBS buffer solution for three times, sucking out residual liquid, changing into DMEM containing 2% fetal calf serum, infecting for 24 hr, and collecting cell supernatant (1mL storing at-70 deg.C)For later TCID50 experiment preparation), washing cells with PBS buffer solution for three times, sucking up residual liquid, adding 2 x protein loading to collect cell samples, boiling for 15min at 96 ℃ in a metal bath, and carrying out Western blot detection. The sophorae ear polysaccharide is found to reduce the expression of the porcine pseudorabies virus variant XJ5 gB protein (A in figure 3), and the fact that the sophorae ear polysaccharide reduces the adsorption and cell entry of the porcine pseudorabies virus variant XJ5 is proved.

(2) TCID50 determination of Sophora polysaccharide on PK-15 cells for inhibiting the adsorption and cell-entering of porcine pseudorabies virus variant XJ 5:

after being digested by Vero cell pancreatin, the Vero cell is diluted by DMEM nutrient solution of fetal bovine serum with volume concentration of 8 percent and is diluted by 2 multiplied by 10³The concentration of each well was added dropwise to a 96-well plate and the plate was placed at 37 ℃ in 5% CO₂After the cells adhere to the wall to form a monolayer and are in a logarithmic growth phase (about 16 hours), the cells are washed for three times by PBS buffer solution, after residual liquid is completely sucked, virus liquid with different concentrations diluted by serum-free DMEM is added, each concentration is repeated for 4 times, after 1.5 hours of infection, the cells are maintained by changing into the DMEM containing 2% fetal calf serum, and the condition of cytopathic effect is observed after 72 hours of infection. The sophorae ear polysaccharide is found to reduce the virus titer of the infected supernatant of the porcine pseudorabies virus variant XJ5 strain (B in figure 3), and the adsorption and cell-entering of the porcine pseudorabies virus variant XJ5 strain are proved to be reduced by the sophorae ear polysaccharide.

(3) INDIRECT Immunofluorecence Assay (IFA) determination of Sophora fruticosa polysaccharide on PK-15 cells inhibits the adsorption and encytosis of porcine pseudorabies virus variant XJ5 strain:

PK-15 cells were digested with trypsin and then diluted with 10% fetal bovine serum by volume in DMEM medium at 5X 10⁵The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO₂After the cells are adhered to form a monolayer and are positioned after the logarithmic growth phase (about 17 hours) in the incubator, the cells are washed for three times by PBS buffer solution at 4 ℃, residual PBS is absorbed out, pagodatree ear polysaccharide (25 mu g, 50 mu g,100 mu g and 200 mu g) and porcine pseudorabies virus variant strain XJ5 which are diluted to corresponding concentrations by 1mL of serum-free DMEM at 4 ℃ are added, the cells are incubated for 1 hour with PK-15 cells at 4 ℃, then 2mL of DMEM nutrient solution containing 2% fetal calf serum is replaced and placed at 37 ℃ and 5% CO₂Incubating in incubator for 1 hr, and storing with corresponding concentration of trametes robiniophila polysaccharide (25 μ g, 50 μ g,100 μ g, 200 μ g)At 37 deg.C, 5% CO₂Culturing in an incubator, washing for three times by using citric acid, washing for three times by using PBS buffer solution, sucking up residual liquid, changing into DMEM containing 2% fetal calf serum, infecting for 24 hours, removing supernatant, washing for three times by using the PBS buffer solution, sucking up residual PBS buffer solution for the last time, adding 4% paraformaldehyde capable of covering the cell surface, fixing for 15 minutes at 37 ℃, penetrating cells for 10 minutes by using 0.1% TritonX-100, washing for three times by using the PBS buffer solution, sucking up for the last time, adding 5% BSA confining liquid for 1.5 hours at 37 ℃ or washing for three times by using the PBS buffer solution after overnight at 4 ℃, sucking up for the last time, and mixing with PRV-resistant pig positive serum 1: 200 dilutions were incubated at 37 ℃ for 2h, washed three times with PBST, blotted out for the last time, and incubated with 1: incubating the 200 diluted secondary goat anti-pig IgG for 1h at 37 ℃, washing for three times by using DAPI (deoxyribose nucleic acid) under a dark condition, completely sucking in the last time, observing cell morphology and pathological changes (C in figure 3) under a fluorescence microscope, and confirming that the pagoda ear polysaccharide reduces the adsorption and cell invasion of the porcine pseudorabies virus variant XJ5 strain.

Example 4

(1) Western blot detection shows that pagodatree ear polysaccharide inhibits the adsorption of porcine pseudorabies virus variant XJ5 strain on PK-15 cells:

PK-15 cells were digested with trypsin and then diluted with a DMEM nutrient solution containing fetal bovine serum at a volume concentration of 5% to 5X 10⁵The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO₂After the cells are adhered to form a monolayer and are positioned after the logarithmic growth phase (about 17 hours) in the incubator, the cells are washed for three times by PBS buffer solution at 4 ℃, residual PBS is absorbed out, pagodatree ear polysaccharide (25 mu g, 50 mu g,100 mu g and 200 mu g) and porcine pseudorabies virus variant strain XJ5 which are diluted to corresponding concentrations by 1mL of serum-free DMEM at 4 ℃ are added, the cells are incubated for 1 hour with PK-15 cells at 4 ℃, then 2mL of DMEM nutrient solution containing 2% fetal calf serum is replaced and placed at 37 ℃ and 5% CO₂Culturing in an incubator, after infecting for 24h, collecting cell supernatant (1mL is stored at-70 ℃ for preparing later TCID50 experiment), washing the cells for three times by PBS buffer solution, sucking up residual liquid, adding 2 x protein loading to collect cell samples, boiling for 15min at 96 ℃ in a metal bath, and carrying out Western blot detection. The discovery that pagodatree ear polysaccharide reduces porcine pseudocerasusExpression of rabies virus variant XJ5 gB protein (A in FIG. 4), confirming that the pagodatree ear polysaccharide reduces the adsorption of porcine pseudorabies virus variant XJ 5.

(2) TCID50 determination of Sophora japonica ear polysaccharide on PK-15 cells for inhibiting adsorption of porcine pseudorabies virus variant XJ 5:

after being digested by Vero cell pancreatin, the Vero cell is diluted by DMEM nutrient solution of fetal bovine serum with volume concentration of 8 percent and is diluted by 2 multiplied by 10³The concentration of each well was added dropwise to a 96-well plate and the plate was placed at 37 ℃ in 5% CO₂After the cells adhere to the wall to form a monolayer and are in a logarithmic growth phase (about 16 hours), the cells are washed for three times by PBS buffer solution, after residual liquid is completely sucked, virus liquid with different concentrations diluted by serum-free DMEM is added, each concentration is repeated for 4 times, after 1.5 hours of infection, the cells are maintained by changing into the DMEM containing 2% fetal calf serum, and the condition of cytopathic effect is observed after 72 hours of infection. It was found that the sophorae ear polysaccharide reduced the virus titer of the infected supernatant of the porcine pseudorabies virus variant XJ5 strain (B in FIG. 4), confirming that the sophorae ear polysaccharide reduced the adsorption of the porcine pseudorabies virus variant XJ5 strain.

(3) INDIRECT Immunofluorecence Assay (IFA) determination of inhibition of adsorption of Sophora auricular polysaccharide on PK-15 cells by the porcine pseudorabies virus variant XJ5 strain:

PK-15 cells were digested with trypsin and then diluted with a DMEM nutrient solution containing fetal bovine serum at a volume concentration of 5% to 5X 10⁵The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO₂After the cells are adhered to form a monolayer and are positioned after the logarithmic growth phase (about 17 hours) in the incubator, the cells are washed for three times by PBS buffer solution at 4 ℃, residual PBS is absorbed out, pagodatree ear polysaccharide (25 mu g, 50 mu g,100 mu g and 200 mu g) and porcine pseudorabies virus variant strain XJ5 which are diluted to corresponding concentrations by 1mL of serum-free DMEM at 4 ℃ are added, the cells are incubated for 1 hour with PK-15 cells at 4 ℃, then 2mL of DMEM nutrient solution containing 2% fetal calf serum is replaced and placed at 37 ℃ and 5% CO₂Culturing in incubator, after infecting for 24h, washing with PBS buffer solution for three times, sucking up residual liquid, adding 4% paraformaldehyde capable of covering cell surface, fixing at 37 deg.C for 15min, penetrating cell with 0.1% TritonX-100 for 10 min, washing with PBS buffer solution for three times, sucking up at last time, adding 5% BSA blocking solution at 37 deg.C for 1.5h or washing with PBS buffer solution for 4 deg.C overnightAll the last time, blotted out, and combined with primary anti-PRV porcine positive serum 1: 200 dilutions were incubated at 37 ℃ for 2h, washed three times with PBST, blotted out for the last time, and incubated with 1: incubating the 200 diluted secondary antibody goat anti-pig IgG for 1h at 37 ℃, dyeing by DAPI for 5min under the condition of keeping out of the sun, washing by PBST for three times, completely sucking the solution for the last time, observing cell morphology and pathological changes under a fluorescence microscope (C in figure 4), and confirming that the pagoda ear polysaccharide reduces the adsorption of the porcine pseudorabies virus variant XJ5 strain.

Example 5

(1) Western blot detection shows that pagodatree ear polysaccharide influences the cell entry of the porcine pseudorabies virus variant XJ5 strain on PK-15 cells.

PK-15 cells were digested with trypsin and then diluted with 10% fetal bovine serum by volume in DMEM medium at 5X 10⁵The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO₂After the cells adhered to form a monolayer in the incubator are positioned after the logarithmic growth phase (about 17 hours), the cells are washed for three times by PBS buffer solution at 4 ℃, after residual PBS buffer solution is absorbed out, 1mL of serum-free DMEM at 4 ℃ and porcine pseudorabies virus variant XJ5 strain are added, after the cells are incubated for 1 hour with PK-15 cells at 4 ℃, the cells are washed for three times by PBS, and the pagodatree ear polysaccharide (25 mu g and 50 mu g) diluted to corresponding concentration by 1mL of serum-free DMEM and PK-15 cells at 37 ℃ and 5 percent CO are added₂After incubation for 1h, washing with citric acid for three times, washing with PBS buffer solution for three times, sucking up the residual liquid, changing to 2mL of DMEM nutrient solution containing 2% fetal calf serum, placing at 37 deg.C and 5% CO₂Culturing in an incubator, infecting for 24h, collecting cell supernatant (1mL, storing at-70 ℃ for later TCID50 experiment preparation), adding 2 x protein loading to collect cell sample, boiling in a metal bath at 96 ℃ for 15min, and performing Western blot detection. It was found that the sophorae ear polysaccharide did not affect the expression of the porcine pseudorabies virus variant XJ5 gB protein (A in FIG. 5), and the results obtained from A in FIG. 5 were subjected to a gray scale analysis (B in FIG. 5), confirming that the sophorae ear polysaccharide affected the encytosis of the porcine pseudorabies virus variant XJ5 at 25. mu.g/ml and 50. mu.g/ml.

(2) TCID50 determination of influence of pagodatree ear polysaccharide on PK-15 cells on cell entrance of porcine pseudorabies virus variant XJ5

Digesting Vero cell pancreatin and then using 8% volume concentration fetal calf bloodDiluted in clear DMEM nutrient solution at 2X 10³The concentration of each well was added dropwise to a 96-well plate and the plate was placed at 37 ℃ in 5% CO₂After the cells adhere to the wall to form a monolayer and are in a logarithmic growth phase (about 16 hours), the cells are washed for three times by PBS buffer solution, after residual liquid is completely sucked, virus liquid with different concentrations diluted by serum-free DMEM is added, each concentration is repeated for 4 times, after 1.5 hours of infection, the cells are maintained by changing into the DMEM containing 2% fetal calf serum, and the condition of cytopathic effect is observed after 72 hours of infection. It was found that the pagodatree ear polysaccharide affects the virus titer of the infected supernatant of the porcine pseudorabies virus variant XJ5 strain at 25. mu.g/ml and 50. mu.g/ml (C in FIG. 5), confirming that the pagodatree ear polysaccharide affects the encytosis of the porcine pseudorabies virus variant XJ5 strain at 25. mu.g/ml and 50. mu.g/ml.

(3) Inject Immunofluorecence Assay (IFA) determination that pagodatree ear polysaccharide does not affect the invasion of porcine pseudorabies virus variant XJ5 strain on PK-15 cells

PK-15 cells were digested with trypsin and then diluted with 10% fetal bovine serum by volume in DMEM medium at 5X 10⁵The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO₂After the cells adhered to form a monolayer in the incubator are positioned after the logarithmic growth phase (about 17 hours), the cells are washed for three times by PBS buffer solution at 4 ℃, after residual PBS buffer solution is absorbed out, 1mL of serum-free DMEM at 4 ℃ and porcine pseudorabies virus variant XJ5 strain are added, after the cells are incubated for 1 hour with PK-15 cells at 4 ℃, the cells are washed for three times by PBS, and the pagodatree ear polysaccharide (25 mu g and 50 mu g) diluted to corresponding concentration by 1mL of serum-free DMEM and PK-15 cells at 37 ℃ and 5 percent CO are added₂After incubation for 1h, washing with citric acid for three times, washing with PBS buffer solution for three times, sucking up the residual liquid, changing to 2mL of DMEM nutrient solution containing 2% fetal calf serum, placing at 37 deg.C and 5% CO₂Culturing in an incubator, after infecting for 24h, removing supernatant, washing for three times by using PBS buffer solution, completely sucking residual PBS buffer solution for the last time, adding 4% paraformaldehyde capable of covering the cell surface, fixing for 15min at 37 ℃, then penetrating cells by using 0.1% TritonX-100 for 10 min, then washing for three times by using PBS buffer solution, completely sucking for the last time, adding 5% BSA blocking solution for 1.5h at 37 ℃ or overnight at 4 ℃, then washing for three times by using PBS buffer solution, completely sucking for the last time, and mixing with PRV-resistant pig positive serum 1: 200 dilution and incubation at 37 deg.C for 2h, usePBST was washed three times, blotted up for the last time, and mixed with 1: 200 diluted secondary goat anti-pig IgG is incubated for 1h at 37 ℃, protected from light, dyed with DAPI for 5min, washed with PBST for three times, blotted up for the last time, and placed under a fluorescence microscope to observe cell morphology and lesion (D in figure 5), thereby confirming that the pagodatree ear polysaccharide affects the cell entry of the porcine pseudorabies virus variant XJ5 strain at 25 mu g/ml and 50 mu g/ml.

Example 6

PK-15 cells were digested with trypsin and then diluted with 10% fetal bovine serum by volume in DMEM medium at 5X 10⁵The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO₂After the cells adhered to the monolayer in the incubator were in the logarithmic growth phase (about 17 hours), the cells were washed three times with PBS buffer, after the residual PBS buffer was aspirated, 1mL of serum-free DMEM was added and the porcine pseudorabies virus variant strain XJ5 was infected, the mixture was placed at 37 ℃ with 5% CO₂After incubation for 1h in an incubator, washing with PBS buffer solution for three times and sucking up residual liquid, adding DMEM 2mL containing 2% fetal calf serum and having sophorae aurin polysaccharide (25 mug, 50 mug, 100 mug and 200 mug) with corresponding concentration, collecting cell samples at 4h and 6h after infection, then carrying out Western blot detection, and carrying out grey scale analysis on the result. As a result, as shown in FIGS. 6 and 7, it was found that the polysaccharide of Sophora japonica affects the expression of porcine pseudorabies virus gB protein, confirming that the polysaccharide of Sophora japonica affects the replication of porcine pseudorabies virus variant XJ5 strain.

Example 7

(1) Western blot detection shows that pagodatree ear polysaccharide is possibly combined with cell surface receptors on PK-15 cells to play a role in preventing infection of porcine pseudorabies virus variant XJ5 strain

PK-15 cells were digested with trypsin and then diluted with 10% fetal bovine serum by volume in DMEM medium at 5X 10⁵The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO₂After the cells adhered to the monolayer in the incubator were in the logarithmic growth phase (about 17 hours), the cells were washed three times with PBS buffer, after the residual PBS buffer was aspirated, 1mL of empty DMEM with the corresponding concentration of pagodatree ear polysaccharide (25. mu.g, 50. mu.g, 100. mu.g, 200. mu.g) was added, and the mixture was placed at 37 ℃ under 5% CO₂Culturing in incubator, infecting pig pseudomorpha after 1hMutant rabies virus XJ5 strain, incubated at 37 ℃ with 5% CO₂After incubation for 1h in the incubator, the cells were collected 24h after infection with DMEM 2ml containing 2% fetal bovine serum, and Western blot detection was performed. The results are shown in fig. 8, and the sophorae ear polysaccharide is found to inhibit the expression of porcine pseudorabies virus gB protein, confirming that sophorae ear polysaccharide may bind to cell surface receptors to play a preventive role.

Preferably 200. mu.g/ml is the preferred amount, depending on the toxicity and related properties of the pagodatree ear polysaccharide.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.