CN109745334B - Application of cordycepin in preparation of medicine for preventing foot-and-mouth disease virus infection - Google Patents
Application of cordycepin in preparation of medicine for preventing foot-and-mouth disease virus infection Download PDFInfo
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- CN109745334B CN109745334B CN201910181159.0A CN201910181159A CN109745334B CN 109745334 B CN109745334 B CN 109745334B CN 201910181159 A CN201910181159 A CN 201910181159A CN 109745334 B CN109745334 B CN 109745334B
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Abstract
The invention relates to an application of cordycepin in preparation of a medicine for preventing foot-and-mouth disease virus infection, belonging to the technical field of veterinary medicines. The application of the invention can provide a high-efficiency, safe and quality-controllable anti-foot-and-mouth disease virus medicine for further controlling the spread of the foot-and-mouth disease.
Description
Technical Field
The invention relates to the technical field of veterinary medicines, and in particular relates to application of cordycepin in preparation of a medicine for preventing foot-and-mouth disease virus infection.
Background
Foot-and-mouth disease virus is a non-enveloped single-stranded positive-strand RNA virus of the picornaviridae family. It has been found that the virus comprises 7 serotypes, I, O, C, asian I, and south africa I, ii, iii, and each serotype is divided into a plurality of subtypes. Foot-and-mouth disease caused by the virus mainly infects animals with cloven hooves such as pigs, cattle and the like, and often forms blisters in the mouth, nose, hooves and other parts, accompanied by clinical symptoms such as fever, trekking and the like. Foot-and-mouth disease has multiple transmission ways, wide epidemic range and strong infectivity, is frequently outbreak in multiple countries at present, seriously threatens the development of the global animal husbandry and has great influence on the world economy and the human society. The disease is listed as the first of a type A animal epidemic disease list by the world animal health organization, China also ranks the disease at the first of a type A animal infectious disease list, and is also one of three single diseases in the China middle and long term animal epidemic disease prevention and treatment plan (2012-2020). At present, vaccine immunization is the main means for preventing and controlling foot-and-mouth disease, however, the use of the vaccine has an "immune window period", i.e. it cannot provide protection for animals within 7 days. Therefore, in order to compensate for the "immune window period", the development of novel effective antiviral drugs is urgently needed.
Disclosure of Invention
The invention aims to provide application of Cordycepin (Cordycepin) in preparing a medicament for preventing foot-and-mouth disease virus infection. The application can provide a high-efficiency, safe and quality-controllable anti-foot-and-mouth disease virus medicine for further controlling the spread of the foot-and-mouth disease.
The invention provides an application of cordycepin in preparing a medicament for preventing foot and mouth disease virus infection.
Preferably, the foot-and-mouth disease virus includes a type a foot-and-mouth disease virus and a type O foot-and-mouth disease virus.
Preferably, the concentration of cordycepin in the medicament is more than 800 [ mu ] mol/L.
The invention provides an application of cordycepin in preparing a medicament for preventing foot and mouth disease virus infection. Cordycepin has inhibitory effect on cell pathological changes induced by A type foot-and-mouth disease virus and O type foot-and-mouth disease virus, and inhibits replication of virus. Cell tests show that the cordycepin has low cytotoxicity and has an inhibiting effect on replication of A type foot-and-mouth disease virus and O type foot-and-mouth disease virus; further experiments confirm that cordycepin only acts early in FMDV replication and does not prevent viral replication when it enters late stage of viral replication. Cordycepin can be used as an effective foot and mouth disease virus resisting component.
Drawings
FIG. 1 is a graph of the cytotoxicity of Cordycepin against IBRS-2 cells at various concentrations in example 1 of the present invention;
FIG. 2 is a graph showing the inhibitory effect of Cordycepin at various concentrations on type O FMDV infection of IBDS-2 cells in example 1 of the present invention;
FIG. 3 is a graph showing the inhibitory effect of Cordycepin at various concentrations on type A FMDV infected IBDS-2 cells in example 1 of the present invention;
FIG. 4 is a graph showing the effect of Cordycepin at different concentrations in example 1 of the present invention on FMDV mRNA inhibition in type O FMDV infected IBDS-2 cells;
FIG. 5 is a graph showing the inhibitory effect of Cordycepin at different concentrations in example 1 of the present invention on FMDV VP1 protein in type O FMDV infected IBDS-2 cells;
FIG. 6 is a graph showing the effect of IFA detection of Cordycepin at different concentrations on the inhibition of FMDV protein expression in type-O FMDV infected cells in example 1 of the present invention;
FIG. 7 is a graph showing the effect of q-PCR detection of Cordycepin on mRNA inhibition in FMDV adsorption and penetration stages in example 1 of the present invention;
FIG. 8 is a graph showing the inhibitory effect of Cordycepin on the expression level of FMDV mRNA of virus-infected cells at different time periods in example 1 of the present invention;
FIG. 9 is a graph showing the inhibitory effect of Cordycepin on the expression level of VP1 protein in virus-infected cells at different time periods in example 1 of the present invention.
Detailed Description
The invention provides an application of cordycepin in preparing a medicament for preventing foot and mouth disease virus infection. In the present invention, the foot-and-mouth disease virus includes a type a foot-and-mouth disease virus and a type O foot-and-mouth disease virus. Cordycepin has inhibitory effect on cell pathological changes induced by A type foot-and-mouth disease virus and O type foot-and-mouth disease virus, and inhibits virus replication, and cordycepin only plays a role in the early replication stage of FMDV and cannot prevent virus replication when entering the later replication stage of virus. The source of the cordycepin is not specially limited, and the cordycepin can be obtained by adopting conventional commercial products in the field.
In the present invention, the concentration of cordycepin in the medicament is preferably > 800. mu. mol/L.
The application of cordycepin in the preparation of a medicament for preventing foot and mouth disease virus infection is further described in detail with reference to specific examples, and the technical scheme of the invention includes but is not limited to the following examples.
Example 1
1. Experimental Material
1.1 cells, viruses and drugs
IBRS-2 cells were preserved from this group; FMDV (O/MY98/BY/2010 and A/GDMM/CHA/2013) is preserved and provided BY the national foot-and-mouth disease reference laboratory; cordycepin was purchased from MCE and formulated in DMSO.
1.2 reagents
DMEM, fetal bovine serum FBS, trypsin medium were purchased from Gibco; MTS assay kit was purchased from Abcam corporation; TRIZOL was purchased from Invitrogen; SYBR Premix Ex TaqⅡThe kits are purchased from precious bioengineering (Dalian) Co., Ltd; RIPA lysate, BCA method protein quantification kit, SDS-PAGE gel preparation kit and ECL from Biyuntian company; BSA, PVDF membranes were purchased from BioRad; tween-20 fromMarine bioengineering, Inc.; triton X-100, DMSO was purchased from Sigma; mouse anti-beta-actin polyclonal antibody, HRP marked anti-rabbit or anti-mouse IgG antibody are purchased from Abcam company; the rabbit anti-O type FMDV VP1 polyclonal antibody is a gift offered by Zhenghai doctor in national foot and mouth disease reference laboratory; the rabbit anti-O type FMDV hyper-immune serum is a gift in China foot and mouth disease reference laboratory.
2. Experimental methods and results
2.1 toxicity test of Cordycepin on IBRS-2 cells:
the cytotoxicity of Cordycepin against IBRS-2 cells was determined using the MTS method. After the cells are paved on a 96-well plate IBRS-2 and fully grow into a monolayer, the culture solution on the upper layer of the cells is discarded, the cells are washed for 3 times by fresh DMEM, finally, 100 mu L of Cordycepin which is diluted by the DMEM culture solution containing 2% FBS in a gradient manner is added, the corresponding DMSO concentration of the preparation solution of the Cordycepin is used as a negative control hole, and the cells are used as cell control holes without any treatment. The cells were incubated at 37 ℃ for 72 hours, the supernatant cell culture was discarded, washed three times with fresh DMEM, and 100. mu.L of fresh DMEM was added, and 20. mu.L of MTS solution was added to each well. After incubation at 37 ℃ for 4h, the absorbance at 490nm was measured on a microplate reader, according to the formula "cell activity rate ═ ODMedicine-ODBlank space)/(ODNegative of-ODBlank space) X 100% "the toxicity of Cordycepin against IBRS-2 cells was calculated at different concentrations. The experiment was independently repeated three times.
The experimental results are shown in fig. 1: MTS results show that the activity rate of cells is still above 80% with the continuous increase of the concentration of the drug, which indicates that the Cordycepin has extremely low cytotoxicity to IBRS-2.
2.2 evaluation of Cordycepin Activity against foot-and-mouth disease Virus on IBRS-2 cells:
well-grown IBRS-2 cells on DMEM complete medium containing 10% FBS were plated in 96-well plates, and after the IBRS-2 cells grew to a full monolayer, the cell supernatant was discarded, washed 3 times with fresh DMEM, and inoculated with FMDV O/MY98/BY/2010(MOI ═ 1). After 1h, virus solution was removed, washed 3 times with fresh DMEM, 100. mu.L Cordycepin diluted in gradient with 2% FBS-containing DMEM was added, virus control wells were prepared with Cordycepin at the corresponding DMSO concentration, and no Cordycep was addedin, no virus as cell control wells. The cells were incubated at 37 ℃ for 24h, the supernatant cell culture was discarded, washed three times with fresh DMEM, and 100. mu.L of fresh DMEM was added, and 20. mu.L of MTS solution was added to each well. After incubation at 37 ℃ for 4h, the absorbance at 490nm was measured on a microplate reader, according to the formula "cell activity rate ═ ODMedicine-ODBlank space)/(ODNegative of-ODBlank space) X 100% "the antiviral effect of Cordycepin at different concentrations was calculated. At the same time, different groups of supernatants were collected, and mRNA of FMDV 2B gene and FMDV VP1 protein levels were detected by q-PCR and Western Blot, respectively. RNA of the cells was extracted according to TRIZOL instructions, and fluorescent quantitative PCR was performed according to SYBR Premix Ex Taq II instructions, and β -actin was used as an internal reference gene. The primer sequence for detecting the specificity of FMDV 2B gene mRNA is as follows:
FMDV-for,5’-CAACAAAACACGGACCCGAC-3’(SEQ ID NO.1)；
FMDV-rev,5’-TTGTACCAGGGTTTGGCCTC-3’(SEQ ID NO.2)；
the primer sequence of beta-actin is as follows:
β-actin for,5’-GACCACCTTCAACTCGATCA-3’(SEQ ID NO.3)；
beta-actin-rev, 5'-GTGTTGGCGTAGAGGTCCTT-3' (SEQ ID NO. 4). The reaction system is as follows: SYBRPremix ExTaq 12.5. mu.L, upstream primer: 1 μ L, downstream primer: 1 μ L, cDNA: 1 μ L, sterilized water: 9.5. mu.L, the reaction program is: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 56 for 30s, and extension at 72 ℃ for 30s, for 40 cycles. According to 2-△△CTThe method calculates the expression level of the sample relative to the reference gene. Extracting protein with protein lysate, and determining the concentration of the extracted protein by using BCA method. Preparing 12% separation gel to carry out protein SDS-PAGE denaturing electrophoresis, and after 2 hours of electrophoresis, electrically transferring the protein to a PVDF membrane. After the membrane is transferred for 2 hours, the membrane is put into 5 percent of freshly prepared skim milk powder for sealing for 1 hour. After blocking, the membrane was placed in rabbit anti-O FMDV VP1 polyclonal antibody (1:3000) and mouse anti-beta-actin polyclonal antibody (1:4000) and incubated overnight in a refrigerator at 4 ℃. Washing the membrane with TBST for 10min 5 times, adding corresponding secondary antibody HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG (1:3000), incubating at room temperature for 1h, washing the membrane with TBST for 5 times, 10min eachFinally, the FMDV VP1 protein is detected by ECL chemiluminescence method. To investigate whether Cordycepin has inhibitory effects on other foot and mouth disease virus subtypes, cells were infected with FMDVA/GDMM/CHA/2013(MOI ═ 1), and their antiviral activities were measured by the MTS method.
The experimental results are shown in FIGS. 2 to 5: the results of examining whether Cordycepin has anti-FMDV virus activity on FMDV by MTS, show that Cordycepin can provide more than 60% protection for IBDV-2 cells (FIG. 2) and inhibit FMDV mRNA expression level (FIG. 4) and VP1 protein expression (FIG. 5) at concentrations of more than 600. mu. mol when different concentrations of drugs are added, respectively. When the concentration is less than 800. mu. mol, Cordycepin does not provide effective protection of cells. Similarly, Cordycepin at concentrations of 600. mu. mol and above was effective in protecting IBRS-2 cells when infected with type A FMDV (FIG. 3), indicating that Cordycepin also has antiviral activity against type A FMDV.
2.3 Indirect immunofluorescence assay for FMDV protein expression in infected cell groups
The density is 3 x 105The/well IBRS-2 cells were plated on a 12-well plate, and after the IBRS-2 cells grew to a monolayer, the cell supernatant was discarded, washed 3 times with fresh DMEM, and inoculated with FMDV O/MY98/BY/2010(MOI ═ 1). After 1h, the virus solution was removed, washed 3 times with fresh DMEM, 100. mu.L of Cordycepin diluted in gradient with 2% FBS-containing DMEM was added, virus control wells were prepared with Cordycepin at the corresponding DMSO concentration, and incubated at 37 ℃ for 12 h. Discarding the upper cell culture solution, washing with PBS for 2 times, fixing cells with 4% paraformaldehyde for 15min, discarding paraformaldehyde, adding methanol for 5min, rinsing with PBS for 3 times, 5min each time, adding blocking solution (10% FBS, 0.3% Triton X-100, 89.7% PBS) for blocking for 10min, adding primary antibody (1:100) diluted with blocking solution, incubating at room temperature for 1h, rinsing with PBS for 3 times, 5min each time, adding secondary antibody (1:200) diluted with blocking solution, incubating at room temperature for 1h, rinsing with PBS for 5 times, 5min each time. Finally, adding 300 mu LDAPI into each hole for dyeing, acting for 5min, rinsing with PBS for 2 times, each time for 5min, and observing the result by a fluorescence microscope.
The experimental results are shown in fig. 6: a large amount of specific fluorescence was observed in the virus-infected IBRS-2 cells after the virus infection and virus-infected groups treated with Cordycepin at a concentration of 600. mu. mol or less, while a small amount of fluorescence was observed in IBRS-2 cells of other treated groups. This result further demonstrates that the anti-foot-and-mouth disease virus activity of Cordycepin on IBRS-2 cells is dose dependent.
2.4 detection of the inhibition of the viral replication phase by FMDV
The IBRS-2 cells were plated in 24-well plates and the culture medium was discarded after the cells grew a monolayer. In the adsorption phase of the virus: FMDV virus solution (MOI ═ 1) was added to each well. Then, Cordycepin was added to each well at different concentrations to give final concentrations of 800. mu. mol and 1000. mu. mol, and negative control groups were set up without adding drugs, and the cell plates were placed in a refrigerator at 4 ℃ for 1 hour to allow only viruses to attach to the cell surfaces. Discarding virus solution, washing away unbound virus with DMEM, adding 400 μ L culture solution into each well, repeatedly freezing and thawing in-80 deg.C refrigerator for 3 times, collecting virus solution, centrifuging at 10000r/min for 10min to remove cell debris, and collecting supernatant for detecting mRNA expression level. In the virus penetration phase: adding 200 mu L of FMDV virus solution into each hole, putting the cells into a 37 ℃ incubator for 1h, allowing the viruses to adsorb the cell surfaces, washing away the unbound viruses by using DMEM, then adding 200 mu L of Cordycepin with different concentrations into each hole, enabling the final concentration of the Cordycepin to be 800 mu mol and 1000 mu mol respectively, simultaneously establishing a negative control group without adding the medicines, putting the cell plate into the 37 ℃ incubator for culturing for 1h, removing the medicines, washing away the residual medicines by using DMEM, adding a maintenance solution containing 2 percent of FBS, putting the cell plate into the 37 ℃ incubator for continuously culturing for 24h, and collecting supernatant to detect the mRNA expression level condition.
The results of the experiment are shown in FIG. 7: in the virus adsorption (Attachment) stage, the cells are respectively treated with Cordycepin and FMDV virus solution with different concentrations and are subjected to combined action in an incubator for 1h, and the result shows that the mRNA expression level of the virus is not obviously different from that of an untreated group no matter the cells are treated with the Cordycepin with different concentrations, so that the Cordycepin does not influence the process of adsorbing the FMDV cells. During the viral Entry (Entry) phase, there was no significant difference in mRNA from each drug-treated group compared to the non-drug-treated negative control group, indicating that Cordycepin did not affect the ability of the virus to enter cells.
2.5 evaluation of the inhibition time of Cordycepin on the infection of foot-and-mouth disease Virus into IBRS-2 cells:
well-grown IBRS-2 cells in DMEM complete medium containing 10% FBS were plated in 12-well plates, and after the IBRS-2 cells grew to a full monolayer, the cell supernatant was discarded, washed 3 times with fresh DMEM, and inoculated with FMDV O/MY98/BY/2010(MOI ═ 1). After 1h, the virus solution was removed, washed 3 times with fresh DMEM, and DMEM with 2% FBS was added as 0 h. Cordycepin was added to different wells at 0h, 2h, 4h, 8h, 16h after viral infection to a final concentration of 1000. mu. mol. And meanwhile, a negative control without adding the medicine is set. CO at 37 deg.C2Culturing in a constant-temperature cell culture box for 24 h. Different groups of supernatants were collected and tested for FMDV mRNA and VP1 protein levels by Q-PCR and Western Blot, respectively.
The experimental results are shown in FIGS. 8 to 9: treatment of cells with Cordycepin at various time periods after viral infection showed significant inhibition of FMDV mRNA levels (figure 8) and VP1 protein levels (figure 9) compared to negative controls within 0-8 h of FMDV replication. The inhibitory effect of Cordycepin is not obvious at 16h, which indicates that Cordycepin only plays a role in the early stage of FMDV replication and cannot prevent the replication of viruses when entering the later stage of virus replication.
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.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
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Claims (2)
1. The application of the cordycepin in the preparation of the medicine for preventing foot-and-mouth disease virus infection is characterized in that the foot-and-mouth disease virus is A type foot-and-mouth disease virus and O type foot-and-mouth disease virus.
2. The use according to claim 1, wherein the concentration of cordycepin in the medicament is > 800 μmol/L.
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