Application of febrifugine dihydrochloride in preparation of preparation for resisting porcine epidemic diarrhea virus infection

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of febrifugin dihydrochloride in preparation of a preparation for resisting porcine epidemic diarrhea virus infection. Specifically, the concentration of the febrifuginine dihydrochloride is 10 to 80 nmol / mL; the preparation for resisting porcine epidemic diarrhea virus infection comprises any one of a medicine, a disinfectant and a feed additive. The used dichroa B dihydrochloride belongs to a dihydrochloride form of traditional Chinese medicine extract dichroa B, and has the advantages of small toxic and side effects, high safety and better solubility. The antifebridin dihydrochloride can reduce cytopathy caused by the porcine epidemic diarrhea virus and inhibit the replication effect of the porcine epidemic diarrhea virus on cells, and provides a scientific and reliable theoretical basis for clinical treatment of the porcine epidemic diarrhea. The dichroa B dihydrochloride can also be used as a feed additive, can effectively prevent the epidemic of porcine epidemic diarrhea in pig farms, and reduces the economic loss of breeding diseases in animal husbandry.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an application of febrifugine dihydrochloride in the preparation of a preparation for resisting porcine epidemic diarrhea virus infection. Background Art

[0002] Porcine Epidemic Diarrhea (PED) is a highly contagious acute intestinal disease caused by Porcine Epidemic Diarrhea Virus (PEDV). PEDV can infect pigs of all ages, but piglets are particularly susceptible, with a mortality rate of up to 50% and a maximum mortality rate of 100%. Symptoms are similar to those of porcine transmissible gastroenteritis (TGE), manifested as acute diarrhea, vomiting and dehydration. The disease often occurs in cold seasons, and the 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 (PEDV) is a member of the Alphacoronavirus family (Coronaviridae, order Nidovirales). It is a single-stranded positive-strand RNA virus with an outer envelope and petal-like protrusions. The inner core is composed of RNA and nucleocapsid protein (N), which is a helical structure with a diameter of 9 to 16 nm and a genome length of 28,000 nucleotides (nt). The virus strain was first isolated in Belgium and named coronavirus CV777. Since 1980, PEDV has become prevalent in Asian countries, but little attention has been paid to PEDV due to its low morbidity and mortality. However, since 2010, highly virulent PEDV variants have appeared in China, with a mortality rate of up to 100% in piglets. In recent years, PED has caused huge economic losses to the global pig industry, especially due to the continuous mutation of the PEDV genome and the low effect of inducing mucosal immunity, which has hindered the development of an effective PEDV vaccine. 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 helpful in solving current clinical problems, but also a new treatment option.

[0004] Febrifugine dihydrochloride is the dihydrochloride form of Febrifugine, and its specific structure is shown in Formula I. Its molecular formula is C 16 H 21 Cl 2 N 3 O 3, compared with febrifugalin, a hydrochloric acid group is introduced to increase the solubility and stability of febrifugalin. Febrifugalin is a quinazolinone alkaloid extracted from the roots and leaves of febrifugalin, which has significant antimalarial activity. Current studies have found that febrifugalin dihydrochloride, in addition to having antimalarial activity, also exhibits excellent pharmacological activity in anti-inflammatory and anti-tumor aspects, and its mechanism of action is mainly related to its ability to inhibit the proliferation and apoptosis of tumor cells and inhibit the protein synthesis of malarial parasites. Moreover, current drug development technology can ensure the safety and effectiveness of febrifugalin dihydrochloride. The present invention first discovered that febrifugalin dihydrochloride has an antiviral effect, specifically anti-infection of porcine epidemic diarrhea virus.

[0005] Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an application of fusin dihydrochloride in the preparation of an anti-porcine epidemic diarrhea virus infection preparation in view of the deficiencies in the prior art, so as to solve the problems existing in the above-mentioned prior art. The present invention finds that fusin dihydrochloride can inhibit the replication of porcine epidemic diarrhea virus in cells and effectively inhibit porcine epidemic diarrhea virus infection. Fusin dihydrochloride can be used as an inhibitory 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 caused by breeding diseases in animal husbandry.

[0007] In order to solve the above technical problems, the present invention discloses an application of febrifugine dihydrochloride in the preparation of a preparation for resisting porcine epidemic diarrhea virus infection.

[0008] Specifically, the concentration of the febrifugation agent dihydrochloride is 10 to 80 nmol / mL.

[0009] Specifically, the anti-porcine epidemic diarrhea virus infection preparation includes any one of a drug, a disinfectant and a feed additive.

[0010] Specifically, the febrifugine dihydrochloride achieves the purpose of resisting porcine epidemic diarrhea virus infection by inhibiting the replication of porcine epidemic diarrhea virus in cells.

[0011] Specifically, the febrifugine dihydrochloride inhibits the replication of porcine epidemic diarrhea virus in a dose-dependent manner.

[0012] The invention also discloses the use of febrifugine dihydrochloride in the preparation of an inhibitor for inhibiting the replication of porcine epidemic diarrhea virus in cells.

[0013] Specifically, the concentration of the febrifugation agent dihydrochloride is 10 to 80 nmol / mL.

[0014] Specifically, the inhibitor for inhibiting the replication of porcine epidemic diarrhea virus in cells includes any one of drugs, disinfectants and feed additives.

[0015] Specifically, in some embodiments of the present invention, an in vitro experiment of the effect of fusanlin dihydrochloride on African green monkey kidney cells and porcine epidemic diarrhea virus shows that fusanlin dihydrochloride can effectively inhibit porcine epidemic diarrhea virus infection, and discloses the mechanism by which fusanlin dihydrochloride inhibits epidemic diarrhea virus infection, that is, fusanlin dihydrochloride mainly inhibits the replication of the virus in cells. The above test results prove the application prospect of fusanlin dihydrochloride in the preparation of anti-porcine epidemic diarrhea virus infection preparations.

[0016] Beneficial effects:

[0017] The invention shows through in vitro cell experiments that febrifugine dihydrochloride can effectively inhibit porcine epidemic diarrhea virus infection. The invention adds febrifugine dihydrochloride at different stages of virus infection and finds that febrifugine dihydrochloride mainly inhibits virus replication to cells.

[0018] The advantage of the febrifugine dihydrochloride used in the present invention is that it is a hydrochloride form of a traditional Chinese medicine extract, has high safety, and is more soluble. It can be used as a biological agent for inhibiting porcine epidemic diarrhea virus, or as a feed additive, effectively preventing the continued prevalence of epidemic diarrhea in pig farms and reducing the economic losses caused by breeding diseases in animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0020] Figure 1 This is a graph showing the cytotoxicity of febrifugationin dihydrochloride on Vero cells in Example 1 of the present invention;

[0021] Figure 2 The results of the determination of the effect of physalisine dihydrochloride on PEDV infection in Vero cells in Example 2 of the present invention are shown in Figure 2; A and B are Western blot and TCID 50 The effect of phytosporin dihydrochloride on PEDV infection in Vero cells was determined. C is the effect of phytosporin dihydrochloride on PEDV infection in Vero cells determined by IFA.

[0022] Figure 3 The results of the determination of the effect of physalisine dihydrochloride on PEDV adsorption on Vero cells in Example 3 of the present invention are shown in FIG. A, B, C and D are Western blot, TCID 50, qRT-PCR and IFA results;

[0023] Figure 4 The results of the determination of the effect of physalisine dihydrochloride on the entry of PEDV into Vero cells in Example 4 of the present invention are shown in FIG. A, B, C and D are Western blot, TCID 50 , qRT-PCR and IFA results;

[0024] Figure 5 The effect of fusabine dihydrochloride on the replication of PEDV in Vero cells was determined by qRT-PCR in Example 5 of the present invention;

[0025] Figure 6 TCID is used in Example 6 of the present invention 50 To determine the effect of physalisine dihydrochloride on the release of PEDV in Vero cells;

[0026] Figure 7 The results of the direct effect determination of fusabine dihydrochloride on PEDV virus in Example 7 of the present invention are shown in Figure 1; wherein A and B are Western blot and TCID of the direct effect of fusabine dihydrochloride on PEDV HLJBY activity. 50 result. DETAILED DESCRIPTION

[0027] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0028] Sources of the raw materials used in the following examples: African green monkey kidney cells (Vero cells), preserved in the laboratory and available to those skilled in the art;

[0029] The porcine epidemic diarrhea virus (PEDV) HLJBY strain was isolated and preserved in the laboratory and is available to those skilled in the art;

[0030] The febrifugine dihydrochloride was purchased from MedChemExpress. It should be noted that the present invention has no special requirements on the source of the febrifugine dihydrochloride. It can be a commercial product commonly used in the art or prepared by itself.

[0031] Other materials: FITC-labeled goat anti-swine IgG was purchased from Sigma (catalog number: D5648; F1638); HSUniversal qPCR Master Mix was purchased from Boyi Bio ACE Co., Ltd. (catalog number: BR0014-01); phalloidin-Alexa Fluor 568 was purchased from Invitrogen (catalog number: A12380); 4% cell tissue fixative was purchased from Solebao Biotechnology Co., Ltd. (catalog number: P1110); PEDV N monoclonal antibody and PEDV pig positive serum were provided by our laboratory and can be obtained by personnel in this field; β-actine mouse monoclonal antibody and horseradish peroxidase (HRP)-labeled goat anti-mouse IgG were purchased from Quanshijin Biotechnology Co., Ltd. (catalog number: HC201-01; HS201-01); CCK-8 kit and DAPI staining solution were purchased from Biyuntian Biotechnology Co., Ltd. (catalog number: C0041; C1005).

[0032] The specific steps of each detection method used in the following embodiments are as follows:

[0033] Western blot test: Cell lysate was added to the cell plate, cell samples were collected, and 2× protein loading buffer was added, and heated at 96°C for 15 min. Equal amounts of whole-cell proteins were separated by 10% separation gel electrophoresis. The electrophoresis conditions were: 80V when the sample passed through the concentrated gel and 120V when it passed through the separation gel. After the electrophoresis, the protein was transferred to the NC membrane by wet transfer. Transfer conditions: constant current 200mA at 4°C, transfer 100min. 5% skim milk was used to block the NC membrane at room temperature for 2h. Subsequently, the NC membrane was incubated with PEDV-N and β-actin antibodies and the corresponding horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibodies, respectively. The primary antibody incubation condition was incubated overnight at 4°C, and the secondary antibody incubation condition was incubated at room temperature for 2h. Finally, the exposure solution was prepared, the NC membrane was immersed in the exposure solution, and incubated for about 1min to develop color in the protein gel imaging system.

[0034] TCID 50 Experiment: Vero cells were cultured at 4×10 4 The cells were seeded at a density of 10 cells / well in a 96-well plate and placed in a 5% CO 2 , 37°C incubator for about 12 hours. When the cells grow to about 70% confluence, the virus solution to be tested is diluted 10 times in a gradient to 10 -8After the dilution is completed, it is inoculated in a 96-well plate, and each dilution is repeated 8 times. After the inoculated cells are incubated in a 37°C incubator for 1.5 hours, the cell supernatant is discarded, and the cells are rinsed 3 times with PBS, and DMEM medium containing 2% fetal bovine serum is added. After 72 hours after infection, the cell pathological effect (CPE) is observed under a microscope, and the TCID of the sample to be tested is calculated using the Reed-Muench method. 50 .

[0035] IFA test: discard the supernatant in the cell culture plate, add 4% cell tissue fixative, and fix in a 37°C incubator for 15 minutes. After fixation, discard the fixative in the plate, permeabilize the membrane with 0.1% Triton X-100 at 37°C for 10 minutes, and then block with 5% BSA in a 4°C refrigerator overnight. After blocking, add 1:200 diluted PEDV-N polyclonal antibody and incubate at 37°C for 2 hours. Then, incubate with 1:200 diluted FITC-labeled goat anti-rabbit antibody at 37°C in the dark for 1 hour. After incubation, incubate the cell culture plate with DAPI stain at room temperature in the dark for 5-7 minutes. Finally, observe the fluorescence under an inverted fluorescence microscope and take pictures and record under a 100x magnification microscope. Each of the above steps requires PBS rinsing 3 times, and the liquid is aspirated the last time.

[0036] qRT-PCR detection: The viral genomic RNA was extracted using the precipitation method (Trizol method). The specific steps were as follows: discard the cell culture medium, wash the cells with PBS three times, and aspirate the PBS cleanly in the last wash to avoid affecting the experimental results; add 1 mL of RNA isolater to a 6-well plate so that the cell surface is fully covered with it to ensure the lysis effect, and use a pipette to gently blow the cells; place the cell lysate in a 1.5 mL centrifuge tube, mix it upside down, and let it stand on ice for 5 minutes; add 1 / 5 volume of chloroform to the collected lysate, shake it vigorously for 15 seconds until it becomes an emulsion, let it stand at 4°C for 5 minutes, and then centrifuge it at 11200 rpm at 4°C for 15 minutes; carefully take out the centrifuge tube, at this time the solution in the centrifuge tube is divided into three obvious layers, the upper layer is a colorless aqueous phase, the middle part is white, and the organic layer is red. Carefully aspirate the upper layer of liquid into a new centrifuge tube; then add an equal volume of pre-cooled isopropanol, mix by inverting, then let it stand quietly at 4°C for about 10 minutes, then centrifuge again at 11200rpm at 4°C for 10 minutes. White precipitate can usually be seen at the bottom of the centrifuge tube; carefully discard the supernatant, and then pour in 1mL 75% ethanol (made from Anase-free ddHO). 2O). Gently flick the tube to suspend the precipitate and invert it several times. Let it stand at room temperature for 3 to 5 minutes, then centrifuge it at 11200 rpm at 4°C for 5 minutes and discard the supernatant. Dry the precipitate in a clean and dry ultra-clean bench at room temperature for 2 to 5 minutes. Pay attention to the time during this process. If it is over-dried, it will be difficult to dissolve the RNA in the end. Add appropriate RNase-free ddH 2 O to dissolve the precipitate. If necessary, gently use a liquid transfer gun to blow it several times, and then proceed to the next step after it is completely dissolved. It can be tested immediately or stored at -85 to -65 °C.

[0037] The extracted viral RNA was tested by qRT-PCR. Reverse transcription was performed according to the EasyScript One-Step gDNA Removaland cDNA Synthesis SuperMix kit to convert RNA into cDNA, and then the reaction system was configured according to the HSUniversal qPCR Master Mix kit for qPCR. The reaction conditions were 95°C pre-denaturation for 30s, 95°C denaturation for 10s, and 60°C annealing for 30s for a total of 45 cycles. The primer sequences are: PEDV N gene-F: GAATTCCAAGGGCGAAAAT, PEDV N gene-R: TTTTCGACAAATTCCGCATCT.

[0038] Example 1: Determination of cytotoxicity of phytochrome P-3-nitropropene dihydrochloride

[0039] The determination of the cytotoxicity of fusanone dihydrochloride on VERO cells includes the following steps: VERO cells are cultured at 4×10 4 cells / well were seeded in a 96-well cell plate and placed in a 5% CO 2 , and cultured in a 37°C incubator for about 12 hours. When the cells grow to about 70% confluence, discard the DMEM medium and wash with PBS three times. Dilute shanshanyi dihydrochloride to 50, 100, 150, and 200 nmol / mL concentrations with 2% fetal bovine serum DMEM and treat the cells. After 24 hours of culture, add 10 μL of CCK-8 solution to each well, incubate for 1 hour, and detect the absorbance value at OD 450nm with a microplate reader. Set up 6 replicate wells for each group. The activity of shanshanyi dihydrochloride on VERO cells was evaluated by CCK-8 test. The concentrations of shanshanyi dihydrochloride were 50, 100, 150, and 200 nmol / mL, respectively. The results are shown in the figure. Figure 1As shown, it can be seen that febrifugine dihydrochloride has obvious cytotoxic effect on VERO cells at concentrations of 150 nmol / mL and 200 nmol / mL, so febrifugine dihydrochloride at a concentration of 100 nmol / mL and below was selected for subsequent experiments.

[0040] Example 2: Effect of febrifugation factor dihydrochloride on PEDV infection of VERO cells

[0041] VERO cells were digested with 0.25% trypsin, diluted with DMEM nutrient solution containing 4% fetal bovine serum, and added dropwise to a 6-well plate at a concentration of 5×10 5 The concentration of cells / well was appropriate and the cells were kept at 37°C and 5% CO 2 After the cells were cultured in an incubator until they attached to the wall and formed a monolayer (about 17-18 hours), the original culture medium was discarded, and the cells were washed three times with PBS solution. After the residual liquid was aspirated, 1 mL of serum-free DMEM and corresponding concentrations of febrifugation ethylenediamine dihydrochloride (10, 20, 40, 80 nmol / mL) were added to each well and placed at 37°C, 5% CO 2 After incubation in the incubator for 1 h, the cells were infected with PEDV HLJBY (MOI = 0.1). During the infection process, VERO cells were kept in the corresponding concentrations of febrifugation ethylenediamine dihydrochloride (10, 20, 40, 80 nmol / mL) and placed at 37°C, 5% CO 2 After incubation in the incubator for 1 h, the cells were replaced with 2 mL of DMEM nutrient solution containing 2% fetal bovine serum and corresponding concentrations of febrifugine dihydrochloride (10, 20, 40, 80 nmol / mL) per well and placed at 37°C with 5% CO 2 Cultured in an incubator, 24 h after infection, cell protein samples and cell supernatant were collected for Western blot and TCID 50 Test; the cells treated in the same way were fixed and used for indirect immunofluorescence assay (IFA) test. Western blot, TCID 50 Test and IFA test results Figure 2 As shown in Figure 2, Western blot results showed that 80 nmol / mL fusin dihydrochloride could significantly inhibit the expression of PEDV N protein in PEDV-infected Vero cells; TCID 50 The experiment measured the virus titer in the supernatant of PEDV-infected cells, and the treatment with fusin dihydrochloride significantly reduced the virus titer in the supernatant; the IFA test results confirmed that fusin dihydrochloride inhibited the infection of PEDV in a dose-dependent manner. The above results show that fusin dihydrochloride can inhibit PEDV infection of VERO cells.

[0042] Example 3: Effect of phytosalicylate dihydrochloride on PEDV adsorption on VERO cells

[0043] In order to study the mechanism of antiviral effect of fusin dihydrochloride in PEDV infection, the effect of fusin dihydrochloride on PEDV adsorption on VERO cells was first investigated. The specific operation was as follows: VERO cells were cultured at 2×10 5 cells / well were inoculated in a 12-well plate. When the cell density reached 70% to 80%, the cells were rinsed 3 times with PBS, and 1 mL of pre-cooled DMEM diluted with different concentrations of febrile safflower oil dihydrochloride (10, 20, 40, 80 nmol / mL) was added and inoculated with 0.1 MOI of PEDV. After infection at 4°C for 1 hour, the cells were rinsed 3 times with PBS and replaced with DMEM containing 2% fetal bovine serum to maintain growth. After 24 hours of infection, cell protein samples and cell supernatants were collected for Western blot and TCID 50 The cells treated in the same way were fixed for IFA test, and VERO was treated with the above method; 1 hour after infection, the cells were washed 3 times with PBS, 1 mL of Trizol was added to lyse the cells, and the viral genomic RNA was extracted by precipitation method for qRT-PCR detection of viral RNA copy number. The experimental results are shown in Figure 3 As shown in the figure, the results of Western blot showed that different concentrations of fusin dihydrochloride did not affect the expression of PEDV N protein in PEDV-infected cells; IFA results confirmed that fusin dihydrochloride did not affect the infection of PEDV; the TCID 50 The results also showed that fusin dihydrochloride had no effect on the virus titer in the supernatant; the qRT-PCR test results showed that fusin dihydrochloride did not change the PEDV virus RNA copy number. The above results show that fusin dihydrochloride has no effect on PEDV adsorption to VERO cells.

[0044] Example 4: Effect of physalisine dihydrochloride on PEDV entry into VERO cells

[0045] In order to study the mechanism of antiviral effect of fusin dihydrochloride in PEDV infection, the effect of fusin dihydrochloride on PEDV entry into VERO cells was further studied, including the following steps: VERO cells were cultured at 2×10 5cells / well were inoculated in a 12-well plate. When the cell density reached 70% to 80%, the cells were rinsed 3 times with PBS, 1 mL of pre-cooled DMEM was added and 0.1 MOI of PEDV was inoculated. After infection at 4°C for 1 hour, the cells were rinsed 3 times with PBS, 1 mL of DMEM containing 2% fetal bovine serum and corresponding concentrations of febrifugine dihydrochloride (10, 20, 40, 80 nmol / mL) were added, and after incubation in the incubator for 1 hour, the cells were first washed 3 times with citric acid solution, then washed 3 times with PBS, and replaced with DMEM containing 2% fetal bovine serum to maintain growth. After 24 hours of infection, cell protein samples and cell supernatants were collected for Western blot and TCID 50 The same treated cells were fixed for IFA test. VERO was treated with the above method; 2 hours after infection, the cells were washed 3 times with citric acid solution, then washed 3 times with PBS, and 1 mL of Trizol was added to lyse the cells. The viral genomic RNA was extracted by precipitation method and used for qRT-PCR to detect the viral RNA copy number. The experimental results are shown in Figure 4 As shown in Figure 2, Western blot results showed that changshansu dihydrochloride did not significantly inhibit the expression of viral PEDVN protein during the cell entry stage; TCID 50 The results of the experimental determination of the virus titer in the cell supernatant were consistent with the results of the Western blot experiment; and the results of IFA and qRT-PCR experiments showed that the effect of safflower oil dihydrochloride on the entry of PEDV into the cells was not significant.

[0046] Example 5: Effect of febrifugation 1-hydroxybutyric acid dihydrochloride on PEDV replication

[0047] In order to study the mechanism of febrifugine dihydrochloride in the process of PEDV infection and further study the effect of febrifugine dihydrochloride on PEDV replication, VERO cells were infected with PEDV for 1 h, and then treated with febrifugine dihydrochloride for 3 h and 5 h. Cell samples were collected 4 h and 6 h after infection to detect the number of copies of viral replication. The specific operation was as follows: VERO cells were cultured at 2 × 10 5 cells / well were inoculated in a 12-well plate. When the cell density reached 70% to 80%, the cells were rinsed 3 times with PBS and inoculated with 0.1 MOI of PEDV. After 1 hour of infection, the cells were rinsed 3 times with PBS and replaced with DMEM containing 2% fetal bovine serum to maintain growth. Corresponding concentrations of physostigmine dihydrochloride (10, 20, 40, 80 nmol / mL) were added. After 4 hours and 6 hours of infection, the cells were washed 3 times with PBS, 1 mL of Trizol was added to lyse the cells, and the viral genomic RNA was extracted by precipitation method for qRT-PCR detection of viral RNA copy number. The results of qRT-PCR experiments are shown in the figure. Figure 5As shown, it can be seen that after treating VERO cells with different concentrations of fusin dihydrochloride, the copy number of PEDV decreased in a dose-dependent manner, and the effect was significant, proving that fusin dihydrochloride can significantly inhibit the replication of PEDV in Vero cells.

[0048] Example 6: Effect of febrifugation factor dihydrochloride on the release of PEDV

[0049] In order to investigate the mechanism by which febrifugine dihydrochloride exerts its antiviral effect during PEDV infection and to further investigate the effect of febrifugine dihydrochloride on PEDV release, VERO cells were infected with PEDV (MOI = 0.1) for 1 h, and then the infected cells were treated with febrifugine dihydrochloride (10, 20, 40, 80 nmol / mL). The supernatant and cells were collected 24 h after infection to determine the PEDV virus titer, and the ratio of extracellular and intracellular PEDV virus titers, TCID 50 The experimental ratio results are as follows Figure 6 As shown, it can be seen that treatment of VERO cells with febrifugation-induced sclerotin dihydrochloride does not affect the release of PEDV.

[0050] Example 7: Effect of febrifugine dihydrochloride on the infectious activity of PEDV

[0051] In order to study the mechanism by which fusin dihydrochloride plays an antiviral role in the process of PEDV infection and further study whether fusin dihydrochloride directly affects the infection activity of PEDV, PEDV was first incubated with different concentrations of fusin dihydrochloride at 37°C for 1 hour, and then infected VERO cells. After 24 hours of infection, the cells were collected for detection, including the following steps: different concentrations of fusin dihydrochloride (10, 20, 40, 80 nmol / mL) diluted in DMEM were mixed with 0.1MOI of PEDV and placed in an incubator for incubation for 1 hour. After the incubation, VERO cells were inoculated and placed in an incubator for infection for 1 hour, then rinsed with PBS 3 times and replaced with DMEM containing 2% fetal bovine serum to maintain growth. After 24 hours of infection, cell protein samples were collected for Western blot. Similarly, Vero cells were cultured at 4×10 4 The cells were seeded at a density of 10 cells / well in a 96-well plate and placed in a 5% CO 2 , 37°C incubator for about 12 hours. When the cells grow to about 70% confluence, the virus solution treated with physostigmine dihydrochloride is diluted 10 times in a gradient to 10 -8After the dilution is completed, it is inoculated in a 96-well plate, and each dilution is repeated 8 times. After the inoculated cells are incubated in a 37°C incubator for 1.5 hours, the cell supernatant is discarded, and the cells are rinsed 3 times with PBS, and DMEM medium containing 2% fetal bovine serum is added. After 72 hours after infection, the cell pathological effect (CPE) is observed under a microscope, and the TCID of the sample to be tested is calculated using the Reed-Muench method. 50 Western blot assay and TCID 50 The test results are as follows Figure 7 As shown in A and 7B, it can be seen that febrifugine dihydrochloride cannot directly inhibit the infection activity of PEDV.

[0052] The present invention uses changshan ethylene dihydrochloride to explore its effect on PEDV infection. According to the test results of Examples 1 to 7, changshan ethylene dihydrochloride has significant cytotoxicity only when the concentration is higher than 100nmol / mL, and 80nmol / mL of changshan ethylene dihydrochloride significantly inhibits PEDV infection after treating cells. Secondly. The present invention further verifies that changshan ethylene dihydrochloride significantly inhibits PEDV infection by inhibiting the replication of PEDV in cells. In summary, the present invention provides a new use of changshan ethylene dihydrochloride as a drug for preventing PEDV infection.

[0053] The present invention provides a method for using changshan ethylenediamine dihydrochloride in the preparation of an anti-swine epidemic diarrhea virus infection preparation. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. Application of physostigmine dihydrochloride in the preparation of preparations for preventing porcine epidemic diarrhea virus infection.

2. The use according to claim 1, characterized in that: The concentration of the febrifugine dihydrochloride is 10-80 nmol / mL.

3. The use according to claim 1, characterized in that: The anti-porcine epidemic diarrhea virus infection preparation comprises any one of a drug, a disinfectant and a feed additive.

4. The use according to claim 1, characterized in that: The febrifugine dihydrochloride achieves the purpose of resisting porcine epidemic diarrhea virus infection by inhibiting the replication of porcine epidemic diarrhea virus in cells.

5. The use according to claim 4, characterized in that: The febrifugine dihydrochloride inhibits the replication of porcine epidemic diarrhea virus in a dose-dependent manner.

6. Use of physostigmine dihydrochloride in the preparation of inhibitors for inhibiting the replication of porcine epidemic diarrhea virus in cells.

7. The use according to claim 6, characterized in that: The concentration of the febrifugine dihydrochloride is 10-80 nmol / mL.

8. The use according to claim 6, characterized in that: The inhibitor for inhibiting the replication of porcine epidemic diarrhea virus in cells includes any one of drugs, disinfectants and feed additives.

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