CN110731958B - Application of EGCG in preparation of preparation for preventing and/or treating PRV infection and preparation for preventing and/or treating PRV infection - Google Patents
Application of EGCG in preparation of preparation for preventing and/or treating PRV infection and preparation for preventing and/or treating PRV infection Download PDFInfo
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Abstract
The invention provides application of epigallocatechin gallate in preparation of a preparation for preventing and/or treating porcine pseudorabies virus infection and a preparation for preventing and/or treating porcine pseudorabies virus, and belongs to the technical field of treatment of porcine pseudorabies virus infection, wherein the concentration of epigallocatechin gallate in the medicine is 10-100 mu mol/L. In the invention, the epigallocatechin gallate has a very significant effect on resisting the infection of the porcine pseudorabies virus: the epigallocatechin gallate can reduce cytopathic effect caused by the porcine pseudorabies virus, inhibit the adsorption, cell entry and replication of the porcine pseudorabies virus, and can directly kill the porcine pseudorabies virus, and more importantly, the epigallocatechin gallate can prevent and/or treat mice infected by the porcine pseudorabies virus, thereby providing a scientific and reliable theoretical basis for clinically treating the porcine pseudorabies.
Description
Technical Field
The invention belongs to the technical field of treatment of porcine pseudorabies virus infection, and particularly relates to application of epigallocatechin gallate in preparation of a preparation for preventing and/or treating porcine pseudorabies virus infection and a preparation for preventing and/or treating porcine pseudorabies virus.
Background
The porcine pseudorabies virus is a linear double-stranded DNA molecule, belongs to herpesviridae and porcine herpesviridae, and has stronger resistance to the external environment because the outside of virus particles are wrapped by a capsule membrane. Pseudorabies virus is pantropic, can proliferate in a variety of tissue culture cells, is most sensitive to rabbit and pig kidney cells (including primary I cells and continuous cell lines), and causes significant cytopathic effects. Pigs are the storage host of pseudorabies virus, and sick pigs, virus-carrying pigs and virus-carrying mice are important infection sources of the pseudorabies virus. In the swine herd, the virus is transmitted mainly through nasal secretions, and in addition, milk and semen are possible modes of transmission. After pseudorabies happens to the pigs, the clinical symptoms of the pigs are different due to the age of the pigs in days, and the death rate of piglets within 15 days can reach 100 percent; infertility, miscarriage, etc. in pregnant sows; boars exhibit testicular swelling, atrophy, loss of reproductive function, etc. Causing huge economic loss to the pig industry.
Pseudorabies (PR), also known as AujeszKy's disease, is a disease of co-suffering from various domestic and wild animals caused by porcine herpes virus type 1 (SuHV-1) or Pseudorabies virus (PRV), and is an acute viral infectious disease characterized by fever, severe itching (excluding pigs), respiratory and nervous system diseases, and encephalomyelitis. The disease is present in an explosive epidemic in pigs. Can cause abortion and stillbirth of pregnant sows, sterility of boars, mass death of newborn piglets, dyspnea and growth retardation of fattening pigs and the like, and is one of serious infectious diseases harming the global pig industry. Since 2011, in many areas of China, severe porcine pseudorabies has been outbreaked, and Bartha-K61 vaccine is inoculated in the pig farms, and the new pseudorabies virus variant strain is different from the classical PRV strain, has high pathogenicity to all ages of pigs and shows different degrees of clinical symptoms, which means that: existing vaccines may not be able to cope with current circulating strains of PRV and may not provide complete immune protection. In short, the live pig farming industry faces a significant risk of PRV. More importantly, the host spectrum of the pseudorabies virus is very wide, and except for infecting vertebrates such as pigs, cattle, sheep, dogs, cats and the like, the mutant porcine pseudorabies virus reported in the prior art can infect people. Therefore, prevention and control of PRV infection appears to be particularly urgent and important.
At present, no effective treatment measures for the variant PRV exist in China, and the occurrence of the variant PRV can be controlled only by early prevention and strengthening sanitary management. In order to reduce the economic losses in the swine industry, there is an urgent need to develop effective drugs for the prevention and treatment of mutated PRV.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of epigallocatechin gallate in preparation of a preparation for preventing and/or treating porcine pseudorabies virus infection, wherein the preparation has characteristics of low cost, no side effect, and significant treatment effect.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides application of epigallocatechin gallate in preparation of a preparation for preventing and/or treating porcine pseudorabies virus infection.
Preferably, the epigallocatechin gallate is used for preventing and/or treating porcine pseudorabies by inhibiting porcine pseudorabies virus adsorption, cell entry and replication.
Preferably, the epigallocatechin gallate prevents and/or treats porcine pseudorabies by directly killing the porcine pseudorabies virus.
The invention provides a preparation for preventing and/or treating porcine pseudorabies virus infection, wherein the concentration of epigallocatechin gallate in the preparation is 10-100 mu mol/L.
Preferably, the concentration of epigallocatechin gallate in the preparation is 20-80 mu mol/L.
Preferably, the concentration of epigallocatechin gallate in the preparation is 40-60 mu mol/L.
Preferably, the solvent of the formulation is PBS buffer or DMSO.
Preferably, the concentration of the PBS buffer solution is 0.05-0.15 mol/L.
Preferably, the concentration of the PBS buffer solution is 0.08-0.12 mol/L.
Preferably, the formulation is obtained by dissolving the epigallocatechin gallate in a solvent.
The invention has the beneficial effects that: the invention provides application of epigallocatechin gallate in preparing a preparation for preventing and/or treating porcine pseudorabies virus infection; the epigallocatechin gallate has a very significant effect on resisting the infection of the porcine pseudorabies virus: the epigallocatechin gallate can reduce cytopathic effect caused by the porcine pseudorabies virus, inhibit the adsorption, cell entry and replication of the porcine pseudorabies virus, and can directly kill the porcine pseudorabies virus, thereby providing scientific and reliable theoretical basis for clinical treatment of the porcine pseudorabies. Meanwhile, the epigallocatechin gallate is the most abundant bioactive polyphenol found in the solid green tea extract, and has the advantages of wide medicine source, easily obtained raw materials, low cost and the like; the epigallocatechin gallate is derived from traditional Chinese medicines, is metabolized by animal organisms, has little pollution to the environment and has no toxic or side effect on animals; the epigallocatechin gallate has multiple biological meanings clinically, and has pharmacological effects of resisting oxidation, resisting arteriosclerosis, resisting thrombosis, resisting vascular proliferation, resisting tumor, etc.
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FIG. 1 is a microscopic observation showing the effect of EGCG on cytopathic effect caused by porcine pseudorabies virus infecting PK-15B6 cells;
FIG. 2 Effect of EGCG on porcine pseudorabies virus infection on PK-15B6 cells; wherein, A is Westernblot for measuring the influence of EGCG on porcine pseudorabies virus infection on PK-15B6 cells; b is TCID50 to determine the effect of EGCG on the infection of porcine pseudorabies virus on PK-15B6 cells; c is the influence of EGCG on the porcine pseudorabies virus infection on PK-15B6 cells measured by fluorescent quantitative PCR;
FIG. 3 is a graph showing the effect of EGCG on porcine pseudorabies virus adsorption and entry cells on PK-15B6 cells, wherein A is Western blot to determine the effect of EGCG on porcine pseudorabies virus adsorption and entry cells on PK-15B6 cells; b is TCID50 to determine the effect of EGCG on the adsorption and entry of porcine pseudorabies virus on PK-15B6 cells; c, determining the influence of EGCG on porcine pseudorabies virus adsorption and entry cells on PK-15B6 cells by fluorescent quantitative PCR (polymerase chain reaction);
FIG. 4 shows the effect of EGCG on porcine pseudorabies virus adsorption on PK-15B6 cells; wherein A is Western blot for determining the influence of EGCG on porcine pseudorabies virus adsorption on PK-15B6 cells; b is TCID50 to determine the effect of EGCG on adsorption of porcine pseudorabies virus on Dulac cells; c is the influence of EGCG on the porcine pseudorabies virus adsorption on PK-15B6 cells measured by fluorescent quantitative PCR;
FIG. 5 shows the effect of EGCG on porcine pseudorabies virus entry on PK-15B6 cells; wherein A is Weatern blot for determining the influence of EGCG on porcine pseudorabies virus entry on PK-15B6 cells; b is the influence of EGCG on porcine pseudorabies virus encytosis on PK-15B6 cells measured by fluorescent quantitative PCR; c is TCID50 to determine the effect of EGCG on porcine pseudorabies virus entry on PK-15B6 cells;
FIG. 6 shows a Western blot to determine the effect of EGCG on porcine pseudorabies virus replication on PK-15B6 cells;
FIG. 7 is the direct killing effect of EGCG on porcine pseudorabies virus; wherein A is Western blot for measuring the direct killing effect of EGCG on the porcine pseudorabies virus, and B is TCID50 for measuring the direct killing effect of EGCG on the porcine pseudorabies virus;
fig. 8 is a graph demonstrating the protection rate of EGCG against challenge mice by their survival rate.
Detailed Description
The invention provides application of epigallocatechin gallate in preparation of a preparation for preventing and/or treating porcine pseudorabies virus infection. In the invention, the epigallocatechin gallate has a very significant effect on resisting the infection of the porcine pseudorabies virus: the epigallocatechin gallate can reduce cytopathic effect caused by porcine pseudorabies virus; inhibiting the adsorption, cell entering and replication of the porcine pseudorabies virus; and can directly kill the porcine pseudorabies virus; through in vivo test in mice, epigallocatechin gallate can prevent and/or treat mice infected by porcine pseudorabies virus. The source of the epigallocatechin gallate is not particularly required, and the epigallocatechin gallate can be prepared by conventional commercial products in the field or by self.
The invention also provides a preparation for preventing and/or treating porcine pseudorabies virus infection, wherein the concentration of epigallocatechin gallate in the preparation is 10-100 mmol/L, preferably 20-80 mmol/L, more preferably 40-60 mmol/L, and most preferably 50 mmol/L. In the present invention, the drug is preferably a liquid formulation, and the solvent of the drug is preferably PBS buffer or DMSO; when the solvent is PBS buffer solution, the concentration of the PBS buffer solution is preferably 0.05-0.15 mol/L, more preferably 0.08-0.12 mol/L, and most preferably 0.1 mol/L.
In the present invention, the drug is preferably obtained by dissolving the epigallocatechin gallate in a solvent. The method and time for dissolving are not particularly limited, and sufficient dissolving can be achieved.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A preparation for preventing and/or treating porcine pseudorabies virus infection is prepared by dissolving epigallocatechin gallate in 0.1mol/L PBS buffer solution, wherein the concentration of epigallocatechin gallate is 50 mmol/L.
Example 2
A preparation for preventing and/or treating porcine pseudorabies virus infection is prepared by dissolving epigallocatechin gallate in 0.15mol/L PBS buffer solution, wherein the concentration of epigallocatechin gallate is 40 mmol/L.
Example 3
A preparation for preventing and/or treating porcine pseudorabies virus infection is prepared by dissolving epigallocatechin gallate in 0.1mol/L PBS buffer solution, wherein the concentration of epigallocatechin gallate is 100 mmol/L.
Example 4
A preparation for preventing and/or treating porcine pseudorabies virus infection is prepared by dissolving epigallocatechin gallate in DMSO at a concentration of 60 mmol/L.
Example 5
Effect of Epigallocatechin gallate on porcine pseudorabies Virus-induced cytopathic Effect
PK-15B6 cells (purchased from Chinese veterinary medicine inspection institute) were diluted with a nutrient solution containing 4% fetal bovine serum and counted, and 5X 10 cells were obtained5Cells at a concentration per well were plated in six well plates and placed in a chamber containing 5% CO237 ℃ incubator. After the cells grow to a density of 70-80% (about 16h), the cells are washed twice by PBS solution,the cells are pretreated for 1h by PBS and EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) with different concentrations respectively, then the cells are infected by porcine pseudorabies virus PRV XJ5 strain (0.1MOI), the liquid is changed after 1h, the infection lasts for 24h and the medicine is always present, and the morphological change and the pathological change of the cells are observed under a microscope. As shown in the results of FIG. 1, EGCG can reduce the cytopathic effect caused by porcine pseudorabies virus. It was shown that EGCG may reduce infection with porcine pseudorabies virus.
Example 6
Activity of epigallocatechin gallate on PK-15B6 cells for inhibiting porcine pseudorabies virus infection (Western blot, fluorescent quantitative PCR and TCID50 experiment)
(1) Western blot determination of Activity of epigallocatechin gallate on PK-15B6 cells for inhibiting infection of porcine pseudorabies virus
PK-15B6 cell digestions were diluted with DMEM nutrient solution containing 4% FBS by volume at 5X 105The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO2After the cells adhere to the wall to form a monolayer in the incubator (about 17-18h), the cells are washed for three times by PBS solution, after residual PBS is absorbed, EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) diluted to the corresponding concentration by 1mL of serum-free DMEM is added, after 1h of incubation with PK-15B6 cells at 37 ℃, the cells are infected by porcine pseudorabies virus and EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) with the corresponding concentration exists, the cells are placed at 37 ℃ and 5 percent CO2After incubation in the incubator for 1h, 2mL of DMEM nutrient solution containing 2% FBS was replaced and EGCG (10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M) was maintained at a corresponding concentration and placed at 37 ℃ in 5% CO2Culturing in an incubator, after 24h infection, collecting cell supernatant (1mL is stored at-70 ℃ for later TCID50 experiment preparation), washing with PBS for three times, sucking up residual liquid, adding 2 x protein loading to collect cell samples, boiling in a metal bath at 96 ℃ for 15min, and then carrying out Westernblot detection. EGCG was found to reduce the expression of porcine pseudorabies gE and UL42 proteins (A in FIG. 2), and it was preliminarily confirmed that EGCG reduced infection with porcine pseudorabies virus.
(2) TCID50 determination of Activity of Epigallocatechin gallate on PK-15B6 cells for inhibiting infection by porcine pseudorabies virus
Volume for digestion of Vero cellsDMEM nutrient solution with concentration of 8% FBS, diluted 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 in the incubator, washing the cells for three times by PBS, after absorbing the residual liquid, adding virus liquid with different concentrations diluted by serum-free DMEM, repeating the steps for 8 times at each concentration, after infecting the cells for 1.5h, changing the cells to DMEM containing 2% FBS for maintenance, and observing the pathological condition of the cells after infecting the cells for 72 h. EGCG was found to reduce the virus titer of porcine pseudorabies virus infection supernatant (B in FIG. 2), confirming that EGCG reduces infection with porcine pseudorabies virus.
(3) Activity of epigallocatechin gallate for inhibiting porcine pseudorabies virus infection on PK-15B6 cells by fluorescent quantitative PCR (polymerase chain reaction) determination
PK-15B6 cell digestions were diluted with DMEM nutrient solution containing 4% FBS by volume 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 culture medium to form a monolayer (about 17-18h), the cells were washed with PBS solution for three times, after residual PBS was aspirated, EGCG (10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M) diluted to the corresponding concentration with 1mL of serum-free DMEM was added, the cells were incubated with PK-15B6 for 1h at 37 ℃, cells were infected with porcine pseudorabies virus and EGCG (10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M) at the corresponding concentration was present, and the cells were incubated at 37 ℃ with 5% CO2After incubation in the incubator for 1h, 2mL of DMEM nutrient solution containing 2% FBS was replaced and EGCG (10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M) was maintained at a corresponding concentration and placed at 37 ℃ in 5% CO2Culturing in an incubator, washing with PBS for three times after infecting for 24h, adding 1mL of serum-free DMEM nutrient solution, placing in a refrigerator at-70 ℃, repeatedly freezing and thawing for 3 times, and extracting DNA for real-time fluorescent quantitative PCR detection. The PCR primers used were: gB94F: 5'-ACAAGTTCAAGGCCCACATCTAC-3' (SEQ ID No. 1); gB94R: 5' -GTCCGTGAAGCGGTTCGTGAT (SEQ ID No. 2); gB probe 5'-FAM-ACGTCATCGTCACGACC-TAMRA-3' (SEQ ID No. 3). The 20. mu.L reaction system used contained 0.5. mu.L of DMSO 0.2. mu. L, TaqDNA polymerase, 0.8. mu.L of each of the upstream and downstream primers, 0.4. mu.L of dNTP, 2. mu. L, DNA 2. mu.L of 10 XBuffer, 0.4. mu.L of the probe, and 12.9. mu.L of RNase-free water. Amplification parameters 95 ℃ 600s, 95 ℃ 10s, 62 ℃ 20s for 45 cycles. As a result, EGCG was able to reduce porcine pseudorabies virus nucleic acid as shown in C in FIG. 2Copy number, EGCG demonstrated to reduce infection by porcine pseudorabies virus.
Example 7
Effect of Epigallocatechin gallate on porcine pseudorabies Virus adsorption and engraftment (Western blot, fluorescent quantitative PCR and TCID50 experiments)
(1) Western blot to determine the effect of epigallocatechin gallate on porcine pseudorabies virus adsorption and entry cells on PK-15B6 cells
PK-15B6 cell digestion DMEM nutrient solution with 4% FBS was diluted to the appropriate density at 5X 105The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO2After the cells adhere to each other in the incubator to form a monolayer (about 17-18h), the cells are washed for three times by PBS solution, after residual PBS is absorbed, EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) diluted to the corresponding concentration by 1mL of serum-free DMEM is added, after 1h of incubation with PK-15B6 cells at 37 ℃, the cells are changed into cold serum-free DMEM, the cells are infected with porcine pseudorabies virus at 4 ℃ and exist in EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) at the corresponding concentration, after 1h of incubation, the cells are washed for three times by cold PBS, DMEM1 CG 1mL containing 2% FBS and exist in EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) at the corresponding concentration are added, after 1h of incubation at 37 ℃, PBS is washed for three times by citric acid, then the cells are washed for three times and absorbed, and residual liquid is changed into 2mL of DMEM nutrient solution containing 2% FBS to maintain, 37 ℃ and 5% CO is placed2Culturing in an incubator, after infecting for 24h, collecting cell supernatant (1mL is stored 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 then carrying out Westernblot detection. As a result, as shown in A in FIG. 3, EGCG was found to reduce the expression of porcine pseudorabies virus gE and UL42 proteins, and it was preliminarily confirmed that EGCG reduced the adsorption and entry of porcine pseudorabies virus into cells.
(2) TCID50 determination of the Effect of Epigallocatechin gallate on porcine pseudorabies Virus encytosis on PK-15B6 cells
Vero cell digestion was diluted with 8% FBS 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 in the incubator, the cells are washed for three times by PBS, and after residual liquid is absorbed completely, serum-free DME is addedAnd repeating the virus solution with different concentrations diluted by M for 8 times at each concentration, changing into DMEM containing 2% FBS for maintenance after 1.5h of infection, and observing the condition of cytopathic effect after 72h of infection. As a result, as shown in B in FIG. 3, EGCG was found to reduce the virus titer of porcine pseudorabies virus infection, confirming that EGCG reduces the adsorption and entry of porcine pseudorabies virus into cells.
(3) Fluorescent quantitative PCR (polymerase chain reaction) determination of influence of epigallocatechin gallate on porcine pseudorabies virus adsorption and entry cells on PK-15B6 cells
PK-15B6 cell digestions were diluted with 4% FBS in DMEM nutrient solution at 5X 105The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO2After the cells adhere to each other in the incubator to form a monolayer (about 17-18h), the cells are washed for three times by PBS solution, after residual PBS is absorbed, EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) diluted to the corresponding concentration by 1mL of serum-free DMEM is added, after 1h of incubation with PK-15B6 cells at 37 ℃, the cells are changed into cold serum-free DMEM, the cells are infected with porcine pseudorabies virus at 4 ℃ and exist in EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) with the corresponding concentration, after 1h of incubation, the cells are washed for three times by cold PBS, DMEM1mL of DMEM containing 2% FBS and exist in EGCG (10M, 20M, 50M and 100M) with the corresponding concentration are added, after 1h of incubation at 37 ℃, PBS is washed for three times by citric acid, then the cells are absorbed for three times, the cells are changed into 2mL of DMEM containing 2% FBS, the nutrient solution is maintained, and 5% CO is maintained at 37 DEG2Culturing in an incubator, after infecting for 24h, washing with PBS for three times, completely absorbing, adding serum-free DMEM1mL, placing in a refrigerator at-70 deg.C, repeatedly freezing and thawing for three times, extracting DNA, and using for fluorescent quantitative PCR determination. As a result, as shown in C in FIG. 3, EGCG was found to reduce the porcine pseudorabies virus nucleic acid copy number, confirming that EGCG reduces the porcine pseudorabies virus adsorption and entry into cells.
Example 8
Effect of Epigallocatechin gallate on porcine pseudorabies Virus adsorption (Western blot, fluorescent quantitative PCR and TCID50 experiment)
(1) Western blot determination of influence of epigallocatechin gallate on porcine pseudorabies virus adsorption on PK-15B6 cells
PK-15B6 cell digestions were diluted with 4% FBS in DMEM nutrient solution at 5X 105Hole/holeWas added dropwise to a 6-well plate and the plate was left at 37 ℃ with 5% CO2After the cells adhere to the wall to form a monolayer (about 17-18h), the cells are washed for three times by PBS solution, after residual PBS is absorbed, EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) diluted to the corresponding concentration by 1mL of serum-free DMEM is added, the EGCG and PK-15B6 cells are incubated for 1h at 37 ℃, the EGCG is washed for three times by cold PBS, the cold serum-free DMEM is changed, porcine pseudorabies virus is infected at 4 ℃ and EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) with the corresponding concentration exists, after 1h of incubation, the EGCG is changed to 2mL of DMEM nutrient solution containing 2% FBS for maintenance, the cells are placed at 37 ℃ and 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 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 then carrying out Western blot detection. As a result, as shown in A in FIG. 4, EGCG was found to reduce the expression of porcine pseudorabies gE and UL42 proteins, confirming that EGCG reduces the adsorption of porcine pseudorabies virus.
(2) TCID50 determination of the Effect of Epigallocatechin gallate on porcine pseudorabies Virus adsorption on PK-15B6 cells
Vero cell digestion was diluted with 8% FBS 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 in the incubator, washing the cells for three times by PBS, after absorbing the residual liquid, adding virus liquid with different concentrations diluted by serum-free DMEM, repeating the steps for 8 times at each concentration, after infecting the cells for 1.5h, changing the cells to DMEM containing 2% FBS for maintenance, and observing the pathological condition of the cells after infecting the cells for 72 h. As a result, as shown in B in FIG. 4, EGCG was found to reduce the virus titer of porcine pseudorabies virus infection, confirming that EGCG reduces the adsorption of porcine pseudorabies virus.
(3) Fluorescent quantitative PCR (polymerase chain reaction) determination of influence of epigallocatechin gallate on porcine pseudorabies virus adsorption on PK-15B6 cells
PK-15B6 cell digestion DMEM nutrient solution with 4% FBS was diluted to the appropriate density at 5X 105The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO2After the cells adhere to the wall to form a monolayer (about 17-18h) in the incubator, the cells are washed by PBS solutionAfter the residual PBS was aspirated for three times, EGCG (10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M) diluted to a corresponding concentration with 1mL of serum-free DMEM was added, the cells were incubated at 37 ℃ for 1 hour with PK-15B6, washed three times with cold PBS, replaced with cold serum-free DMEM, infected with porcine pseudorabies virus at 4 ℃ and present with EGCG (10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M) at a corresponding concentration, after incubation for 1 hour, washed with PBS and aspirated, 800. mu.L of PBS was added, the cells were scraped with a cell scraper, a cell sample was collected (without repeated freeze-thaw), DNA was extracted, and fluorescence quantitative PCR was performed. As a result, as shown in C in FIG. 4, EGCG was found to reduce the nucleic acid copy number of porcine pseudorabies virus, confirming that EGCG reduces the adsorption of porcine pseudorabies virus.
Example 9
Effect of Epigallocatechin gallate on porcine pseudorabies Virus encytosis (Western blot, fluorescent quantitative PCR and TCID50 experiments)
(1) Western blot to determine the influence of epigallocatechin gallate on porcine pseudorabies virus entry on PK-15B6 cells
PK-15B6 cell digestion DMEM nutrient solution with 4% FBS was diluted to the appropriate density at 5X 105The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO2After the cells adhere to the wall to form a monolayer in the incubator (about 17-18h), washing the cells for three times by using cold PBS solution, sucking up residual PBS, adding 1mL of serum-free cold DMEM and infecting porcine pseudorabies virus, incubating at 4 ℃ for 1h, washing the cells for three times by using cold PBS solution and sucking up residual liquid, adding 1mL of cold DMEM containing 2% FBS and EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) with corresponding concentration, incubating at 37 ℃ for 1h, washing the cells for three times by using citric acid, then washing the cells for three times by using PBS, sucking up residual liquid, changing the cells into 2mL of DMEM nutrient solution containing 2% FBS for maintenance, placing at 37 ℃ and 5% CO2Culturing in an incubator, after infecting for 24h, collecting cell supernatant (1mL is stored 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 then carrying out Westernblot detection. As a result, as shown in A in FIG. 5, EGCG was found to reduce the expression of porcine pseudorabies gE and UL42 proteins, confirming that EGCG inhibits the encytosis of porcine pseudorabies.
(2) TCID50 determination of the Effect of Epigallocatechin gallate on porcine pseudorabies Virus encytosis on PK-15B6 cells
Vero cell digestion was diluted with 8% FBS 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 in the incubator, washing the cells for three times by PBS, after absorbing the residual liquid, adding virus liquid with different concentrations diluted by serum-free DMEM, repeating the steps for 8 times at each concentration, after infecting the cells for 1.5h, changing the cells to DMEM containing 2% FBS for maintenance, and observing the pathological condition of the cells after infecting the cells for 72 h. As a result, as shown in B in FIG. 5, EGCG was found to reduce the virus titer of porcine pseudorabies virus infection, confirming that EGCG inhibits the entry of porcine pseudorabies virus.
(3) Fluorescent quantitative PCR (polymerase chain reaction) determination of influence of epigallocatechin gallate on porcine pseudorabies virus entry on PK-15B6 cells
PK-15B6 cell digestions were diluted with 4% FBS in DMEM nutrient solution at 5X 105The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO2After the cells adhere to each other in the incubator to form a monolayer (about 17-18h), washing the cells for three times by using cold PBS solution, sucking up residual PBS, adding 1mL of serum-free cold DMEM and infecting porcine pseudorabies virus, incubating at 4 ℃ for 1h, washing for three times by using cold PBS cells and sucking up residual liquid, adding 1mL of DMEM containing 2% FBS and EGCG (10 mu M, 20 mu M, 50 mu M and 100 mu M) with corresponding concentration, incubating at 37 ℃ for 1h, washing for three times by using citric acid, then washing for three times by using PBS, sucking up residual liquid, adding 800 mu L of PBS, scraping the cells by using a cell scraper, collecting cell samples (without repeated freeze thawing), extracting DNA, and performing fluorescence quantitative PCR determination. As a result, as shown in C in FIG. 5, EGCG was found to reduce the nucleic acid copy number of porcine pseudorabies virus, confirming that EGCG inhibits the entry of porcine pseudorabies virus.
Example 10
Effect of Epigallocatechin gallate on replication of porcine pseudorabies Virus (Western blot)
PK-15B6 cell digestions were diluted with 4% FBS in DMEM nutrient solution at 5X 105The concentration of each well was added dropwise to a 6-well plate at 37 ℃ with 5% CO2In the incubator, cells are waited to adhere to the wall to form a monolayerThen (about 17-18h), the cells were washed three times with PBS solution, after the residual PBS was aspirated, 1mL serum-free DMEM was added and the porcine pseudorabies virus was infected, placed at 37 ℃ with 5% CO2After incubation in the incubator for 1h, PBS cells were used three times and the residual liquid was aspirated, DMEM2mL containing 2% FBS and in the presence of EGCG (10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M) at the corresponding concentration, cell samples were collected at 4h, 6h after infection and subjected to Westernblot assay. As a result, as shown in FIG. 6, EGCG was found to reduce the expression of porcine pseudorabies virus gE and UL42 proteins, confirming that EGCG inhibits the replication of porcine pseudorabies virus.
Example 11
Direct killing effect of epigallocatechin-3-gallate on porcine pseudorabies virus
PBS or EGCG (10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M) solution was incubated with PRV at 37 ℃ for 1h, then PK-15B6 cells were inoculated for 1h, washed three times with PBS, changed to 2% DMEM, and after 24h, protein samples were collected and changes in porcine pseudorabies virus protein were detected using western blot. TCID50 is made for the above incubated virus, and the change of porcine pseudorabies virus titer is detected. The results are shown in fig. 7, and the EGCG is found to reduce the virus titer of porcine pseudorabies virus infection, and the EGCG can be proved to kill the porcine pseudorabies virus directly.
Example 12
Effect of Epigallocatechin gallate on porcine pseudorabies Virus infection in mice
Female mice at 6 weeks of age were divided into seven groups: the drug administration method comprises the following steps of (1) 6 healthy control groups (corresponding PBS for intraperitoneal injection), 6 drug administration control groups (high-dose drug for intraperitoneal injection), 6 toxin attacking control groups (corresponding PBS for intraperitoneal injection), 4-day-ahead drug administration groups (6 low-dose groups and 6 high-dose groups), 2-day-ahead drug administration groups (6 low-dose groups and 6 high-dose groups), and 1-hour drug administration groups (6 low-dose groups and 6 high-dose groups) after toxin attack. The concentration of EGCG was 20mg/ml, the low dose was 20mg/kg per mouse and the high dose was 40mg/kg per mouse. The toxic dose of PRV is 1X104TCID50, injecting EGCG into abdominal cavity of mice in administration group and 4 days earlier before challenge, and simultaneously administering corresponding amount of PBS to other mice for 4 days and 4 days later; the group with 2 days ahead of the administration is administered in the abdominal cavity 2 days before the toxicity is attackedThe medicine is administered for 2 days, and then the medicine is administered for 2 days; the administration group is administered 1h after the toxin is attacked, and the administration is performed 1h after the toxin is attacked, and the administration is continuously performed for 4 days. After challenge, mice were counted as dead per day for each group. As a result, it was found that all of the mice in the challenge group died on day 7, 4 survived, 2 died, 3 survived and 3 died in the groups administered 4 days earlier (low dose) and 2 days earlier (low dose), 3 survived in the group administered 1h after challenge (low dose), and all survived in the groups healthy, drug-administered, 4 days earlier (high dose), 2 days earlier (high dose) and 1h after challenge (high dose) (fig. 8). The EGCG (40mg/kg) is shown to provide complete protection for the porcine pseudorabies virus infection, and the EGCG (40mg/kg) is found to have prevention and treatment effects on the porcine pseudorabies virus infection.
As can be seen from the above examples, the epigallocatechin gallate has a very significant effect of resisting porcine pseudorabies virus infection, can reduce cytopathic effect caused by porcine pseudorabies virus, inhibit the adsorption, cell entry and replication of porcine pseudorabies virus, and can directly kill porcine pseudorabies virus, and more importantly, epigallocatechin gallate can prevent and/or treat mice infected with porcine pseudorabies virus.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (10)
1. Application of epigallocatechin gallate in preparing a preparation for preventing and/or treating variant porcine pseudorabies virus infection, wherein epigallocatechin gallate reduces cytopathic effect caused by porcine pseudorabies virus; and/or, reducing the expression of porcine pseudorabies virus gE and UL42 proteins; and/or, reducing viral titer of porcine pseudorabies virus infection; and/or, reducing the porcine pseudorabies virus nucleic acid copy number;
wherein the variant porcine pseudorabies virus strain is porcine pseudorabies virus PRV XJ5 strain.
2. The use according to claim 1, wherein the epigallocatechin gallate is used for preventing and/or treating porcine pseudorabies by inhibiting porcine pseudorabies virus adsorption, entry and replication.
3. The use according to claim 1, wherein the epigallocatechin gallate prevents and/or treats porcine pseudorabies by directly killing porcine pseudorabies virus.
4. The use according to claim 1, wherein the concentration of epigallocatechin gallate in the preparation is 10 to 100 μmol/L.
5. The use according to claim 4, wherein the concentration of epigallocatechin gallate in the preparation is 20-80 μmol/L.
6. The use according to claim 4, wherein epigallocatechin gallate is present in the formulation at a concentration of 40 to 60 μmol/L.
7. The use according to claim 4, wherein the formulation uses a solvent which is PBS buffer or DMSO.
8. The use of claim 7, wherein the concentration of the PBS buffer is 0.05-0.15 mol/L.
9. The use of claim 8, wherein the concentration of the PBS buffer is 0.08-0.12 mol/L.
10. The use according to any one of claims 5 to 9, wherein the preparation is obtained by dissolving epigallocatechin gallate in a solvent.
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| CN113648324B (en) * | 2021-06-28 | 2022-09-16 | 扬州大学 | Medicine, disinfectant and additive for resisting porcine pseudorabies virus and preparation method thereof |
| CN114277082B (en) * | 2021-12-09 | 2023-06-16 | 河南省农业科学院动物免疫学重点实验室 | Method for inhibiting pseudorabies virus in-vitro replication and proliferation by using inhibitor A2ti-1 and application |
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| CN102949387A (en) * | 2011-08-19 | 2013-03-06 | 格鲁吉亚健康科学大学研究所公司 | Composition for treating herpes simplex viruses (HSVs), and method thereof |
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| EGCG debilitates the persistence of EBV latency by reducing the DNA binding potency of nuclear antigen 1;Ya-Lin Chen等;《Biochemical and Biophysical Research Communications》;20111227;第417卷;1093-1099 * |
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| Herpes simplex virus virucidal activity of MST-312 and epigallocatechin gallate;Prajakta Pradhan和Marie L. Nguyen;《Virus Research》;20180328;第249卷;93-98 * |
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