CN109745328A - A kind of application of homoharringtonine in the drug of preparation prevention mouth disease virus infection - Google Patents
A kind of application of homoharringtonine in the drug of preparation prevention mouth disease virus infection Download PDFInfo
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- CN109745328A CN109745328A CN201910181171.1A CN201910181171A CN109745328A CN 109745328 A CN109745328 A CN 109745328A CN 201910181171 A CN201910181171 A CN 201910181171A CN 109745328 A CN109745328 A CN 109745328A
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- homoharringtonine
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Abstract
The present invention relates to application of the homoharringtonine in the drug of preparation prevention mouth disease virus infection, belong to veterinary drug technical field.Application of the present invention can provide a kind of efficient, safe and quality controllable foot-and-mouth disease virus resistant drug for the propagation of further control aftosa.
Description
Technical field
The present invention relates to veterinary drug technical fields, and in particular to a kind of homoharringtonine prevents hoof-and-mouth disease in preparation
Application in the drug of poison infection.
Background technique
Foot and mouth disease virus is a kind of no coating single strand plus RNA virus of a kind of Picornaviridae.Have now found that the virus
Including 7 kinds of serotypes such as A type, I, II, III types of O-shaped, c-type, Asia I type and South Africa, while each serotype is divided into multiple hypotypes again.
The aftosa as caused by the virus, the artiodactyls such as main infection pig, ox often form bubble and companion at positions such as mouth, nose, hoof
With fever, the clinical symptoms such as row of crossing mountains.Aftosa route of transmission is more, Epidemic Scope is wide and infectiousness is strong, at present in multiple country's frequencies
It is numerous to break out, the development of global animal husbandry is seriously threatened, and tremendous influence is caused to world economy and human society.It is by generation
Boundary's animal health tissue is classified as first of A class animal epidemic list, which is also come the of a kind of zoonosis list by China
One, while being also one of three single-row diseases in " long-term animal epidemic control program (2012-2020) in country ".Mesh
Before, vaccine immunity is the main means of prevention and control aftosa, however i.e. it cannot be at 7 days for the use of vaccine presence " immune window phase "
Within to animal provide protection.Therefore, in order to make up " immune window phase ", it is badly in need of developing novel effective antiviral drugs.
Summary of the invention
The purpose of the present invention is to provide homoharringtonines (Homoharringtonine) to prevent hoof-and-mouth disease in preparation
Application in the drug of poison infection.The application can be provided for the propagation of further control aftosa it is a kind of efficiently, safety and
Quality controllable foot-and-mouth disease virus resistant drug.
The present invention provides application of the homoharringtonine in the drug of preparation prevention mouth disease virus infection.
Preferably, the foot and mouth disease virus includes A type foot and mouth disease virus and O-shaped foot and mouth disease virus.
Preferably, 6.2 μm of ol/L of concentration > of the homoharringtonine in the drug.
The present invention provides application of the homoharringtonine in the drug of preparation prevention mouth disease virus infection.High tricuspid
The cytopathy that China fir ester alkali induces A type foot and mouth disease virus and O-shaped foot and mouth disease virus has inhibiting effect, inhibits answering for virus
System.Test cell line shows that homoharringtonine cytotoxicity is low, equal to the duplication of A type foot and mouth disease virus and O-shaped foot and mouth disease virus
It is inhibited；Further it is experimentally confirmed that homoharringtonine only works in FMDV duplication early stage, and enter viral
The duplication of virus cannot be then prevented when replicating the later period.Homoharringtonine can as a kind of effective foot-and-mouth disease virus resistant at
Point.
Detailed description of the invention
Fig. 1 is cytotoxicity of the Homoharringtonine to IBRS-2 cell of various concentration in the embodiment of the present invention 1
Figure；
Fig. 2 is that the Homoharringtonine of various concentration in the embodiment of the present invention 1 is thin to O-shaped FMDV infection IBRS-2
The inhibiting effect figure of born of the same parents；
Fig. 3 is that the Homoharringtonine of various concentration in the embodiment of the present invention 1 is thin to A type FMDV infection IBRS-2
The inhibiting effect figure of born of the same parents；
Fig. 4 is the Homoharringtonine of various concentration in the embodiment of the present invention 1 to O-shaped FMDV infection cell
FMDV mRNA inhibiting effect figure；
Fig. 5 is VP1 of the Homoharringtonine to O-shaped FMDV infection cell of various concentration in the embodiment of the present invention 1
Protein expression inhibiting effect figure；
Fig. 6 is that the Homoharringtonine that IFA detects various concentration in the embodiment of the present invention 1 infects carefully O-shaped FMDV
The FMDV protein expression inhibiting effect figure of born of the same parents；
Fig. 7 is that q-PCR detection Homoharringtonine adsorbs FMDV, penetrates in the stage in the embodiment of the present invention 1
MRNA inhibiting effect figure；
Fig. 8 is for Homoharringtonine in the embodiment of the present invention 1 in different time sections to the FMDV of virus infected cell
MRNA inhibiting effect figure；
Fig. 9 is for Homoharringtonine in the embodiment of the present invention 1 in different time sections to the VP1 of virus infected cell
Protein expression inhibiting effect figure.
Specific embodiment
The present invention provides application of the homoharringtonine in the drug of preparation prevention mouth disease virus infection.In this hair
In bright, the foot and mouth disease virus includes A type foot and mouth disease virus and O-shaped foot and mouth disease virus.Homoharringtonine is to A type hoof-and-mouth disease
The cytopathy of malicious and O-shaped foot and mouth disease virus induction has inhibiting effect, inhibits the duplication of virus, and homoharringtonine only exists
FMDV duplication early stage works, and the duplication of virus cannot be then prevented when the entrance virus replication later period.The present invention is to high cepehalotaxus fortunei
The source of ester alkali does not have special restriction, using this field conventional commercial product.
In the present invention, concentration of the homoharringtonine in the drug is preferably 6.2 μm of ol/L of >.
Foot and mouth disease virus is prevented in preparation to a kind of homoharringtonine of the present invention combined with specific embodiments below
Application in the drug of infection is further described in detail, and technical solution of the present invention includes but is not limited to following embodiment.
Embodiment 1
1. experimental material
1.1 cells, virus and drug
IBRS-2 cell is saved by this seminar；FMDV (O/MY98/BY/2010 and A/GDMM/CHA/2013) is by country
Aftosa reference laboratory is saved and is provided；Homoharringtonine is purchased from MCE company, is prepared with DMSO.
1.2 reagent
DMEM, fetal calf serum FBS, trypsase culture medium are purchased from Gibco company；MTS detection kit is public purchased from Abcam
Department；TRIZOL is purchased from Invitrogen company；II kit of SYBR Premix Ex Taq is purchased from precious bioengineering (Dalian)
Co., Ltd；RIPA lysate, BCA method protein quantification kit, PAGE gel reagent preparation box, ECL are purchased from the green skies
Company；BSA, pvdf membrane are purchased from BioRad company；Tween-20 is purchased from Shanghai Sheng Gong bio-engineering corporation；Triton X-100,
DMSO is purchased from Sigma company；Anti- β-actin the polyclonal antibody of mouse, HRP label anti-rabbit or dynamics are purchased from Abcam
Company；The O-shaped FMDV VP1 polyclonal antibody of rabbit-anti is given by national aftosa reference laboratory doctor Zheng Haixue；Rabbit-anti is O-shaped
FMDV hyper-immune serum is given by national aftosa reference laboratory Zhou Guangqing.
2. experimental method and result
Toxicity test of the 2.1Homoharringtonine on IBRS-2 cell:
Using MTS method measurement Homoharringtonine to the cytotoxicity of IBRS-2 cell.Wait be taped against 96 orifice plates
After IBRS-2 cell grows full single layer, cell culture supernatants are discarded, are washed 3 times with fresh DMEM, is eventually adding with containing 2%
The good 100 μ L of Homoharringtonine of the DMEM culture solution gradient dilution of FBS, with the preparation of Homoharringtonine
The corresponding DMSO concentration of solution is as negative control hole, to make cell control well with no treatment.It is put into 37 DEG C of lasting cultures
72h discards upper cell culture solution, is washed with fresh DMEM and adds the fresh DMEM of 100 μ L three times, and 20 μ L are added in every hole
MTS solution.37 DEG C are incubated for the absorbance value surveyed in microplate reader at 490nm after 4h, according to formula " cell activity rate=(ODDrug-
ODBlank)/(ODIt is negative-ODBlank) × 100% " calculates toxicity of the Homoharringtonine to IBRS-2 cell of various concentration.
Experiment is independent in triplicate.
Experimental result as shown in Figure 1: MTS the results show that being continuously increased with drug concentration, the activity rate of cell still exist
85% or more, illustrate that Homoharringtonine is extremely low to IBRS-2 cytotoxicity.
2.2Homoharringtonine active evaluation of foot-and-mouth disease virus resistant on IBRS-2 cell:
IBRS-2 cell well-grown on DMEM complete medium containing 10%FBS is taped against 96 orifice plates, to IBRS-2
After cell grows full single layer, cell culture supernatants are discarded, are washed 3 times with fresh DMEM, is inoculated with 100TCID50O/MY98/BY/
2010.After 1h, remove virus liquid, washed 3 times with fresh DMEM, it is good with the DMEM culture solution gradient dilution containing 2%FBS to be added
100 μ L of Homoharringtonine, using the corresponding DMSO concentration of the preparation solution of Homoharringtonine as virus control
Cell control well with no Homoharringtonine, virus-free is made in hole.It is put into 37 DEG C and persistently cultivates 48h, it is thin to discard upper layer
Born of the same parents' culture solution is washed with fresh DMEM and adds the fresh DMEM of 100 μ L three times, and 20 μ L MTS solution are added in every hole.37 DEG C incubate
The absorbance value at 490nm is surveyed in microplate reader after educating 4h, according to formula " cell activity rate=(ODDrug-ODBlank)/(ODIt is negative-
ODBlank) × 100% " calculates the antivirus action of various concentration Homoharringtonine.It is collected simultaneously different groups of supernatants
Liquid, q-PCR and Western Blot detect mRNA the and FMDVVP1 protein level of FMDV 2B gene respectively.It is said according to TRIZOL
Bright book extracts the RNA of cell, carries out quantitative fluorescent PCR according to II operational manual of SYBR Premix Ex Taq, β-actin makees
For reference gene.For detecting the special primer sequence of FMDV 2B gene mRNA are as follows:
FMDV-for,5'-CAACAAAACACGGACCCGAC-3'(SEQ ID NO.1)；
FMDV-rev,5'-TTGTACCAGGGTTTGGCCTC-3'(SEQ ID NO.2)；
The primer sequence of β-actin are as follows:
β-actin for,5'-GACCACCTTCAACTCGATCA-3'(SEQ ID NO.3)；
β-actin-rev,5'-GTGTTGGCGTAGAGGTCCTT-3'(SEQ ID NO.4).Reaction system are as follows: SYBR
Premix ExTaq:12.5 μ L, upstream primer: 1 μ L, downstream primer: 1 μ L, cDNA:1 μ L, aqua sterilisa: 9.5 μ L, response procedures
Are as follows: 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 5s, 56 annealing 30s, 72 DEG C of extension 30s, 40 recycle.According to 2-△△CTMethod calculates
Expression quantity of the sample relative to reference gene.Albumen is extracted with protein lysate, the protein concentration extracted using the measurement of BCA method.
The separation gel for preparing 12% carries out protein SDS-PAGE denaturing electrophoretic, and after electrophoresis 2h, albumen electricity is transferred on pvdf membrane.Transferring film
After 2h, 5% skimmed milk power of film merging Fresh is carried out closing 1h.It is after closing, film merging rabbit-anti is O-shaped
FMDV VP1 polyclonal antibody (1:3000), in the anti-β-actin polyclonal antibody (1:4000) of mouse, 4 DEG C of refrigerators are incubated overnight.
It is washed film 5 times, each 10min with TBST, film is put into corresponding secondary antibody HRP later and marks goat anti-rabbit igg, HRP marks goat anti-
Mouse IgG (1:3000) is incubated at room temperature 1h, and TBST is washed film 5 times, each 10min, finally utilizes the development inspection of ECL chemoluminescence method
Survey FMDV VP1 albumen.There is antiviral activity in order to study Homoharringtonine to A type foot and mouth disease virus, utilize
100TCID50A/GDMM/CHA/2013 infection cell, MTS method measure its antiviral activity.
Experimental result is as shown in Figure 2-5: whether having antiviral work to FMDV with MTS detection Homoharringtonine
Property, under the drug condition for being separately added into various concentration as the result is shown when concentration is 6.2 μm of ol and concentrations above,
Homoharringtonine can just provide IBRS-2 cell and (Fig. 2) is effectively protected, and significantly inhibit FMDV mRNA
With the expression (Fig. 4, Fig. 5) of VP1 protein level.And when concentration is lower than 6.2 μm of ol, Homoharringtonine cannot then be mentioned
It is effectively protected for cell.Equally, when with A type Foot-and-mouth disease virus Infectious Cycle, 6.2 μm of ol and concentrations above
IBRS-2 cell can be also effectively protected in Homoharringtonine, illustrate that Homoharringtonine also has A type FMDV
There is biological activity (Fig. 3).
The expression of FMDV albumen in 2.3 indirect immunofluorescene assay infection cell groups
It is 3 × 10 by density5/ hole IBRS-2 cell is taped against 12 orifice plates, after IBRS-2 cell grows full single layer, discards thin
Born of the same parents' culture supernatants are washed 3 times with fresh DMEM, are inoculated with 100TCID50O/MY98/BY/2010.After 1h, remove virus liquid, uses
Fresh DMEM is washed 3 times, and Homoharringtonine 100 μ good with the DMEM culture solution gradient dilution containing 2%FBS is added
L is put into 37 DEG C of lasting cultures using the corresponding DMSO concentration of the preparation solution of Homoharringtonine as virus control wells
12h.Upper cell culture solution is discarded, PBS is cleaned 2 times, and the fixed cell 15min of 4% paraformaldehyde discards paraformaldehyde, is added
Methanol acts on 5min, is rinsed 3 times, each 5min with PBS, addition confining liquid (10%FBS, 0.3%Triton X-100,
10min 89.7%PBS) is closed, the primary antibody (1:100) that addition later has been diluted with confining liquid is incubated at room temperature 1h, PBS rinsing 3
Secondary, each 5min, the secondary antibody (1:200) that addition has been diluted with confining liquid is incubated at room temperature 1h, and PBS is rinsed 5 times, each 5min.Most
Every hole is added 300 μ LDAPI and is dyed afterwards, acts on 5min, and PBS is rinsed 2 times, each 5min, fluorescence microscope result.
Experimental result is as shown in Figure 6: IBRS-2 cell after untreated virus infection and with 0.1,1.5,3.1 μm of ol
The visible a large amount of specificity fluorescent of the processed infection group and viral infection group of Homoharringtonine, and the IBRS-2 of other processing groups
Then there is a small amount of fluorescence in cell.This result further confirms Homoharringtonine on IBRS-2 cell
Dose dependent is presented in foot-and-mouth disease virus resistant activity.
The detection in 2.4FMDV suppressing virus replication stage
IBRS-2 cell is spread into 24 orifice plates, after cell covers with single layer, discards culture solution.In the absorption rank of virus
Section: FMDV virus liquid is added in every hole.Then the Homoharringtonine of various concentration is added into every hole, makes its final concentration
Respectively 6.2,12.5,25 μm of ol, set up the negative control group for being added without drug, cell plates are put into 4 DEG C of refrigerator effect 1h, only
Virus is allowed to be attached to cell surface.Virus liquid is discarded, unbonded virus is washed away with DMEM, 400 μ L culture solutions are added in every hole,
It is put into -80 DEG C of refrigerator multigelations 3 times, collects virus liquid, 10000r/min is centrifuged 10min and removes cell fragment, and supernatant is used
In detection mRNA expression situation.Penetrate the stage in virus: the FMDV virus liquid of 200 μ L is added in every hole, and cell is put into 37
DEG C incubator 1h allows virus absorption onto cell surface, unbonded virus is washed away with DMEM, various concentration then is added into every hole
200 μ L of Homoharringtonine, making the final concentration of Homoharringtonine is respectively 6.2,12.5,25 μm of ol, together
When set up the negative control group that drug is not added, cell plates are put into 37 DEG C of incubator culture 1h, removes drug, washes away residual with DMEM
Drug, be added the maintaining liquid containing 2%FBS, be put into 37 DEG C of incubators continue cultivate 48h, collect supernatant detection mRNA express water
Flat situation.
Experimental result is as shown in fig. 7, in absorption (Attachment) stage of virus, respectively with various concentration
Homoharringtonine and FMDV virus liquid handles cell, in the incubator collective effect 1h, no matter uses difference as the result is shown
The Homoharringtonine of concentration handles cell, viral mRNA expression and untreated fish group without significant difference, so
Homoharringtonine will not have an impact the absorption of FMDV.(Entry) stage, each drug-treated are penetrated in virus
The mRNA of group compares no significant difference with the negative control group without drug-treated, illustrates that Homoharringtonine will not
Influence the ability of cell entry cell.
2.4Homoharringtonine inhibits the evaluation of time to mouth disease virus infection IBRS-2 cell:
IBRS-2 cell well-grown on the DMEM complete medium containing 10%FBS is taped against 12 orifice plates, to IBRS-
After 2 cells grow full single layer, cell culture supernatants are discarded, are washed 3 times with fresh DMEM, is inoculated with 100TCID50O/MY98/
BY/2010.After 1h, remove virus liquid, washed 3 times with fresh DMEM, is added with the DMEM culture solution containing 2%FBS, at this time conduct
0h.Homoharringtonine is added into different holes respectively in 0h after the virus infection, 2h, 4h, 8h, 16h, keeps its end dense
Degree is 12.5 μm of ol.The negative control that drug is not added is set up simultaneously.37℃CO248h is cultivated in constant temperature cell incubator.It collects not
With group supernatant, q-PCR and Western Blot detects mRNA the and FMDVVP1 protein level of FMDV 2B gene respectively.
Experimental result is as shown in Fig. 8~9: different time sections Homoharringtonine is handled thin after the virus infection
Born of the same parents, the results show that in 0~8h of FMDV duplication, compared with negative control, FMDV mRNA level in-site (Fig. 8) and VP1 albumen water
Dawn shows and is suppressed (Fig. 9).And in 16h, the inhibiting effect of Homoharringtonine is not obvious, explanation
Homoharringtonine only works in FMDV duplication early stage, and cannot then prevent virus when the entrance virus replication later period
Duplication.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>application of a kind of homoharringtonine in the drug of preparation prevention mouth disease virus infection
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
caacaaaaca cggacccgac 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttgtaccagg gtttggcctc 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gaccaccttc aactcgatca 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gtgttggcgt agaggtcctt 20
Claims (3)
1. application of the homoharringtonine in the drug of preparation prevention mouth disease virus infection.
2. application according to claim 1, which is characterized in that the foot and mouth disease virus includes A type foot and mouth disease virus and O-shaped
Foot and mouth disease virus.
3. application according to claim 1, which is characterized in that concentration > of the homoharringtonine in the drug
6.2μmol/L。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11850250B2 (en) | 2020-03-30 | 2023-12-26 | Hangzhou Minsheng Pharmaceutical Co., Ltd. | Use of homoharringtonine in preparation of betacoronavirus replication inhibitor in human |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109419804A (en) * | 2017-08-21 | 2019-03-05 | 中国农业大学 | A kind of plant source preparation of livestock and poultry viral infection resisting |
-
2019
- 2019-03-11 CN CN201910181171.1A patent/CN109745328A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109419804A (en) * | 2017-08-21 | 2019-03-05 | 中国农业大学 | A kind of plant source preparation of livestock and poultry viral infection resisting |
Non-Patent Citations (1)
| Title |
|---|
| GONG, MEI-JIAO等: "Inhibitory effects of homoharringtonine on foot and mouth disease virus in vitro", 《JOURNAL OF MEDICAL VIROLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11850250B2 (en) | 2020-03-30 | 2023-12-26 | Hangzhou Minsheng Pharmaceutical Co., Ltd. | Use of homoharringtonine in preparation of betacoronavirus replication inhibitor in human |
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Application publication date: 20190514 |