CN107308168A - A kind of medicine for suppressing the intracellular duplication of porcine reproductive and respiratory syndrome virus - Google Patents
A kind of medicine for suppressing the intracellular duplication of porcine reproductive and respiratory syndrome virus Download PDFInfo
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Abstract
The invention provides a kind of medicine for suppressing the intracellular duplication of porcine reproductive and respiratory syndrome virus.The present invention have studied the influence that 25 hydroxy cholesterols (25HC) are replicated to porcine reproductive and respiratory syndrome virus (PRRSV) in cellular level, as a result find that 25HC can significantly inhibit the duplication of many strains of PRRSV, and suppress the absorption of PRRSV replicative cycles and enter the stage, without influence it is virus genomic synthesis and virion release, and 25HC to PRRSV virion without direct deactivation.25HC is expected to a kind of drug candidate of the exploitation for preventing and treating PRRS.The present invention provides new medicine for the prevention and treatment of porcine reproductive and respiratory syndrome, with preferable market value and potential applicability in clinical practice.
Description
Technical field
The present invention relates to antiviral drugs field, in particular it relates to which a kind of suppression porcine reproductive and respiratory syndrome virus is thin
The medicine that intracellular is replicated.
Background technology
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,
PRRS it is) by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome
Virus, PRRSV) caused by a kind of communicable disease for seriously endangering global pig industry.The disease with cause sow breeding difficulty and
Each age group respiratory diseases in pigs is characterized.PRRSV is the single strand plus RNA virus for having cyst membrane, belongs to shell type virales artery
Scorching Viraceae Arterivirus, it is different with antigenicity according to hereditary capacity, it is divided into Genotype I and gene II types, virus tool
Have height variation property, its field lasting evolution and variation cause the generation of many recombinant strains and variation strain so that
Increase the complexity and arduousness of PRRS prevention and control.Current prevention and control PRRS main method is vaccine inoculation, but the security of vaccine
There are problems with validity, it is impossible to meet the demand of control and prevention of disease, so it is a kind of new to develop effective antiviral drugs
Thinking.
25-HYDROXY CHOLESTEROL (25-hydroxycholesterol, 25HC) is a kind of hydroxylate of cholesterol, is consolidated by courage
The hydroxylase catalysis cholesterol of alcohol 25 is produced.25HC has various biological function, can adjust lipid metabolism, and induction B cell is migrated,
Suppress the generation of immunoglobulin A (Immunoglobulin A, IgA) and adjust inflammatory reaction.
With going deep into for PRRSV molecular biology researches, there is presently no set for different phase in PRRSV replicative cycles
Count out the report of effective antiviral thing.And medicine antiviral effect in vivo and adverse reaction etc. are also needed further
In vivo studies and clinical test checking.
The content of the invention
It is an object of the invention to provide a kind of medicine for treating porcine reproductive and respiratory syndrome virus.
The present invention is studied by lot of experiments and found, with the biology point for adjusting lipid metabolism, adjusting inflammatory reaction function
Sub- 25-HYDROXY CHOLESTEROL (25-hydroxycholesterol, 25HC), which has, suppresses porcine reproductive and respiratory syndrome virus duplication
Effect.
Therefore the present invention provides a kind of medicine for treating porcine reproductive and respiratory syndrome, contains 25HC.
Further, the present invention provides a kind of medicine for suppressing the intracellular duplication of porcine reproductive and respiratory syndrome virus, should
Contain 25-HYDROXY CHOLESTEROL in medicine.
Further, medicine also contains pharmaceutically acceptable auxiliary material.
Said medicine falls within the protection of the present invention with the pharmaceutical preparation that medically acceptable carrier or excipient are made
Scope.
The preparation is tablet, capsule, powder-injection, spray or granule.
The present invention provides a kind of prevention containing 25-HYDROXY CHOLESTEROL and/or treats the group of porcine reproductive and respiratory syndrome
Compound.
Composition the invention provides 25-HYDROXY CHOLESTEROL or containing it is in the medicine for suppressing PRRSV duplications is prepared
Application.
Preferably, described PRRSV is JXwn06, HB-1/3.9, VR-2332 (MLV), CHsx1401 and GZ11-G1.
It is a discovery of the invention that during 5 μM of concentration, 25HC can make PRRSV strains (HB-1/3.9, CHsx1401 and GZ11-G1)
Virus titer is reduced to 1/100 or 1/1000, and in 10 μM of concentration, 25HC can make PRRSV strains (HB-1/3.9, CHsx1401
And GZ11-G1) virus titer be reduced to 1/10000；For VR-2332 (MLV) strain, in 5 μM of concentration, 25HC can make disease
Malicious titre is reduced to 1/10, and in 10 μM of concentration, 25HC can make virus titer be reduced to 1/1000；For JXwn06 strains,
During 1.25 μM of concentration, 25HC can make virus titer be reduced to 1/10, and in 10 μM of concentration, 25HC can make virus titer be reduced to 1/
100000, and the dose-dependent reduction of rise virus titer presentation with 25HC concentration from 1.25 μM to 10 μM.
Composition the invention provides 25-HYDROXY CHOLESTEROL or containing it suppresses PRRSV in preparation to be replicated in the cell
Medicine in application.
Composition the invention provides 25-HYDROXY CHOLESTEROL or containing it is preparing suppression PRRSV absorption host cells
Medicine in application.
Composition the invention provides 25-HYDROXY CHOLESTEROL or containing it suppresses PRRSV in preparation and enters host cell
Medicine in application.
In above-mentioned pharmacy application, described 25-HYDROXY CHOLESTEROL is to PRRS virion without deactivation.
In above-mentioned pharmacy application, the 25-HYDROXY CHOLESTEROL does not suppress the virus genomic synthesis of PRRS.
In above-mentioned pharmacy application, the 25-HYDROXY CHOLESTEROL does not suppress the release of PRRS virion.
The present invention is tested by indirect immunofluorescence experiment, virus titer measure, cell activity assays, viruses adsorption, disease
Poison enters experiment, viral release test, and real-time fluorescence quantitative RT-PCR finds that 25-HYDROXY CHOLESTEROL can be by suppressing PRRSV
Adsorb and enter cell, and suppress duplication of the virus in host cell.The present invention is further discovered that 25-HYDROXY CHOLESTEROL pair
Virion does not suppress the synthesis of PRRSV genomes and the release of virion without direct deactivation.The present invention is pig breeding
Prevention and treatment with respiration syndrome provide new medicine, with preferable market value and potential applicability in clinical practice.
Brief description of the drawings
Figure 1A-Fig. 1 E is after pretreatment cell, 25HC significantly inhibit the design sketch of PRRSV duplications.Figure 1A：Detect 25HC's
Cytotoxicity；Figure 1B：The thin of PRRSV infection is substantially reduced after indirect immunofluorescene assay, 25HC pretreatment MARC-145 cells
Born of the same parents' number；Fig. 1 C：Virus titer is determined, and PRRSV duplications are significantly inhibited after 25HC pretreatment MARC-145 cells；Fig. 1 D：25HC is pre-
PRRSV duplications are significantly inhibited after processing PAMs；Fig. 1 E：After pretreatment cell, 25HC significantly inhibits answering for different genotype PRRSV
System.
Fig. 2A-Fig. 2 C is first infect virus, then with 25HC processing, 25HC significantly inhibits PRRSV copy patterns.Fig. 2A：25HC
To PRRSV without deactivation.Fig. 2 B and Fig. 2 C：Virus is first infected, then with 25HC processing, 25HC significantly inhibits PRRSV duplications.Disease
Poison is infected after MARC-145 cells (Fig. 2 B) and PAMs (Fig. 2 C) respectively, and with 25HC processing, 25HC is notable in virus infection early stage
Suppress PRRSV to replicate.
Fig. 3 A- Fig. 3 D are the design sketch of absorption and entrance that 25HC can suppress PRRSV.Fig. 3 A and Fig. 3 B：25HC suppresses
PRRSV absorption.In MARC-145 cells (Fig. 3 A) and PAMs (Fig. 3 B), 25HC can suppress PRRSV absorption.Fig. 3 C and
Fig. 3 D：25HC suppresses PRRSV entrance.In MARC-145 cells (Fig. 3 C) and PAMs (Fig. 3 D), 25HC can suppress PRRSV
Entrance.
Fig. 4 A- Fig. 4 C are that 25HC does not suppress the synthesis of PRRSV genomes and the releasing effect figure of virion.Fig. 4 A and figure
4B：25HC does not suppress the synthesis of PRRSV genomes.In MARC-145 cells (Fig. 4 A) and PAMs (Fig. 4 B), 25HC does not suppress
The synthesis of PRRSV genomes.Fig. 4 C show that 25HC does not influence PRRSV release.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention
In the case of essence, the modifications or substitutions made to the inventive method, step or condition belong to the scope of the present invention.
Unless otherwise specified, chemical reagent used in embodiment is skill used in conventional commercial reagent, embodiment
The conventional meanses that art means are well known to those skilled in the art.
MARC-145 cells, porcine alveolar macrophage (the Porcine pulmonary used in the embodiment of the present invention
Alveolar macrophages, PAMs) preserved by applicant.PRRSV strains JXwn06 (Highly pathogenic (HP)-
PRRSV), HB-1/3.9 (Low-pathogenic PRRSV), comes from VR-2332 (Type 2PRRSV) commercialization attenuated live
Vaccine strain (modified-live virus, MLV) is purchased from Boehringer Ingelheim Vetmedica China point public affairs
Department, CHsx1401 (NADC30-like PRRSV) and GZ11-G1 (Type 1PRRSV) are preserved by laboratory.25HC (article No.s
H1015-10MG Sigma-Aldrich companies (St.Louis, MO, USA)) are purchased from.Fluorescein isothiocyanate
(FITC)-conjugated sheep anti-mouse iggs (H+L) (article No. 115-095-003) are purchased from Jackson ImmunoResearch
Laboratories companies (West Grove, PA, USA).RPMI-1640 and DMEM (Dulbecco ' s modified
Eagle ' s medium) it is purchased from Thermo Fisher Scientific companies (Waltham, MA, USA), hyclone
(Fetal bovine serum, FBS) is purchased from Hyclone companies (South Logan, UT, USA).CCK-8 kits (Cell
Counting Kit-8) (article No. C0037) purchased from green skies company (Shanghai, China).The anti-PRRSV N proteins monoclonal antibody of mouse
Prepared by this experiment.Trizol (article No. 206101) is purchased from Magen companies (Beijing, China).SYBR Select Master
Mix (article No. 4472897) is purchased from Thermo Fisher Scientific companies.FastQuant RT Kit(With
GDNase) (article No. KR106-02) is purchased from Tiangeng company (Beijing, China).Absolute ethyl alcohol (Ethanol) (article No. 64-17-5) is purchased
From Chemical Reagent Co., Ltd., Sinopharm Group (Shanghai, China).
In the embodiment of the present invention, absolute ethyl alcohol (Ethanol) is 25HC solvent, is used as solvent control.The setting of control
(ethanol consumption, processing time etc.) is identical with experimental group 25HC setting.
Inhibition tests of the 25HC of embodiment 1 to PRRSV
1st, method
1.1 indirect immunofluorescence assay
With the MARC-145 cells of the absolute ethyl alcohol of precooling after room temperature fixes infection PRRSV 30 minutes (30minutes,
30min), mouse source PRRSV N proteins monoclonal antibody (1:1000 dilutions) as primary antibody, 37 DEG C act on 2 hours (2hours,
2h), then PBS is washed 3 times, FITC-conjugated sheep anti-mouse iggs (H+L) (1:200 dilutions) it is used as secondary antibody, 37 DEG C of effect 1h
Washed 3 times, finally observed with fluorescence microscope (Nikon, Japan) with PBS afterwards.
1.2 virus titers are determined
MARC-145 cells are laid in 96 orifice plates, 48h postoperative infection PRRSV, 48h (48hours post- after infection
Infection, 48hpi) cell is collected, using microtitrimetry and combination indirect immunofluorescence assay measure virus titer, knot
Fruit is with TCID50/ ml (50%tissue culture infective dose per ml) is represented.
1.3 cell activity assays
Using CCK-8 kit detection cells activity.MARC-145 cells are laid on (5 × 10 in 96 orifice plates3Individual cell is every
Hole), cultivate and 25HC is added after 12h, act on 48h, then add CCK-8 solution (10 μ l are per hole), 37 DEG C of effect 1h are finally determined
450nm light absorption value.The kit principle of this experimental applications is to judge cytoactive with light absorption value of the cell at 450nm,
This experiment is to determine whether 25HC has an impact to cytoactive by being compared with solvent control Ethanol, therefore need to compare 25HC
With the difference of Ethanol light absorption values.
1.4 viruses adsorptions are tested
MARC-145 cells and PAMs are laid in 24 orifice plates, 37 DEG C of culture 48h (MARC-145 cells) and 12h (PAMs)
After be transferred to 4 DEG C of environment, place 1h, 25HC (10 μM) and PRRSV mixed liquor inoculating cell 2h then used under the conditions of 4 DEG C, makes disease
Poison is adsorbed onto cell surface without entering, and cell is washed with precooling PBS 3 times after virus infected cell, then adds fresh medium,
Virus titer is detected after 37 DEG C of culture 48h.
1.5 cell entries are tested
MARC-145 cells and PAMs are laid in 24 orifice plates, 37 DEG C of culture 48h (MARC-145 cells) and 12h (PAMs)
After be transferred to 4 DEG C of environment, place 1h, then PRRSV inoculating cells, sense made 2h, make viruses adsorption to cell surface under the conditions of 4 DEG C
Without entering, cell is washed with precooling PBS 3 times after virus infection, then add the fresh medium containing 25HC (10 μM), 37 DEG C
3h is incubated, supernatant is abandoned, cell is washed with precooling PBS 3 times, virus titer is detected after supplementing fresh medium, 37 DEG C of culture 48h.
1.6 viral release tests
MARC-145 cells are laid in 24 orifice plates, inoculation PRRSV (MOI=0.01) after 37 DEG C of culture 48h, 24hpi is abandoned
Supernatant, then add containing 25HC (10 μM) 5%DMEM, 15 after liquid is changed, 30,45 and 60min collects supernatant respectively
Cell simultaneously detects virus titer.
1.7 real-time fluorescence quantitative RT-PCR
Infection PRRSV cell (MARC-145 cells and PAMs) total serum IgE is extracted using Trizol methods, with the RNA of extraction
For masterplate, cDNA is synthesized with FastQuant reverse transcription reagent box.Using cDNA as masterplate, with reference to SYBR Select Master
Mix use condition, using real time fluorescence quantifying PCR method, with endogenous β-actin (MARC-145 cells) or PPIA
(PAMs) as reference gene, the relative amount of PRRSV genomes is detected.Using detection PRRSV non-structural proteins 9
The primer detection PRRSV genomes of (Nonstructural protein, Nsp9) code area, sequence is：：5′-CCT GCA
ATT GTC CGC TGG TTT G-3 ' (sense primer) and 5 '-GAC GAC AGG CCA CCT CTC TTA G-3 ' (downstreams
Primer)；Detect β-actin primer sequence：5 '-TCC CTG GAG AAG AGC TAC GA-3 ' (sense primer) and 5 '-
AGC ACT GTG TTG GCG TAC AG-3 ' (anti-sense primer)；Detect PPIA primer sequence：5′-AAG GTT CCT GCT
TTC ACA GAA TAA T-3 ' (sense primer) and 5 '-AAT TTC TCT CCA TAG ATG GAC TTG C-3 ' (downstreams
Primer).Using 2-ΔΔCTMethod calculates the relative amount of PRRSV genomes.
1.8 data analysis
Test data is represented with mean+SD (means ± standard deviations), uses GraphPad
Two-way ANOVA in Prism (version 5.0, GraphPad Software, San Diego, CA, USA) software
Test statistical methods carry out significance difference analysis to data.P<0.05 (*), represents significant difference；P<0.01 (* *) and P<
0.001 (* * *), represents that difference is extremely notable.
2 results
After 2.1 pretreatment cells, 25HC significantly inhibits PRRSV duplications
PRRSV duplications can be suppressed in order to study 25HC, whether have detected 25HC using CCK-8 kits first right
MARC-145 cells are toxic, as a result show, compared with solvent (absolute ethyl alcohol) control group (25HC=0 μM), various concentrations
25HC (2.5-20 μM) without significantly reducing, shows 25HC at 2.5-20 μM light absorption value of the MARC-145 cells at 450nm
In concentration range, to MARC-145 cytotoxics (see Figure 1A).
Can 25HC suppress PRRSV duplications：MARC-145 cells are pre-processed with the 25HC of various concentrations (1.25-10 μM)
24h, is then inoculated with PRRSV (JXwn06, MOI 0.01), and 48hpi receives sample and detected with indirect immunofluorescence assay, as a result shown,
Without cell positive PRRSV, but in solvent (absolute ethyl alcohol) control group, PRRSV can be replicated Mock groups efficiently in cell.With
Solvent (absolute ethyl alcohol) control group is compared, and the cell number of 25HC treatment group PRRSV infections is substantially reduced, and with 25HC concentration
Rise (1.25-10 μM) PRRSV infection cell number in being gradually reduced trend, this shows that 25HC significantly inhibits answering for PRRSV
Make and in dose dependent (see Figure 1B).In order to further determine that inhibitions of the 25HC to PRRSV, present invention various concentrations
25HC pretreatment MARC-145 cell 24h, then be inoculated with PRRSV (JXwn06, MOI 0.01), 48hpi receive sample, detection virus
Titre, as a result shows, compared with solvent (absolute ethyl alcohol) control group, PRRSV is in MARC-145 cells in 25HC treatment groups
Virus titer is substantially reduced.Wherein, at low concentration (1.25 μM), 25HC can make virus titer be reduced to 1/10, in high concentration
Virus titer can be made to be reduced to 1/100000 when (10 μM), and in dose dependent, this shows that 25HC can significantly inhibit PRRSV and exist
Duplication in MARC-145 cells (see Fig. 1 C).Pre-processed with the 25HC of various concentrations, concentration range is 1.25 to 10 μM, respectively
1.25 μM are taken, 2.5 μM, 5 μM, 10 μM of 4 concentration are tested.
It has detected after pretreatment PAMs, the shadow that 25HC is replicated to PRRSV (JXwn06, the type of gene 2, highly pathogenic strain)
Ring, as a result show, compared with solvent (absolute ethyl alcohol) control group, 25HC can make virus titer be reduced to 1/ at concentration (5 μM)
100, this shows that 25HC can equally significantly inhibit duplications of the PRRSV in PAMs (see Fig. 1 D).
It has detected whether 25HC can suppress different genotype PRRSV duplication, the present embodiment have selected HB-1/3.9 (bases again
Because of 2 types, low pathogenic strain), VR-2332 (MLV, the type vaccine strain of gene 2), CHsx1401 (type of gene 2, low pathogenic strain)
It is representative with 4 strains of GZ11-G1 (type of gene 1), these poison can be suppressed after have detected 25HC pretreatment MARC-145 cells
The duplication of strain, as a result shows, compared with solvent (absolute ethyl alcohol) control group, and in 5 μM of concentration, 25HC can make PRRSV strains (HB-
1/3.9, CHsx1401 and GZ11-G1) virus titer be reduced to 1/100 or 1/1000, in 10 μM of concentration, 25HC can make
The virus titer of PRRSV strains (HB-1/3.9, CHsx1401 and GZ11-G1) is reduced to 1/10000.And for VR-2332
(MLV) strain, in 5 μM of concentration, 25HC can make virus titer be reduced to 1/10, and in 10 μM of concentration, 25HC can make virus titer
It is reduced to 1/1000 (see Fig. 1 E).To sum up, 25HC, which pre-processes MARC-145 cells and PAMs, can significantly inhibit PRRSV duplications, together
When illustrate 25HC may act on cell and play suppress PRRSV replicate function.
After 2.2 PRRSV infections, 25HC remains to significantly inhibit PRRSV duplications
Because it can significantly inhibit PRRSV duplications after 25HC pretreatment cells, in order to study after PRRSV infection 25HC pairs
The influence that PRRSV is replicated, the present embodiment have detected whether 25HC has inactivating efficacy to PRRSV virion, by 25HC and
Then PRRSV (JXwn06, MOI 0.01) detects the virus titer of 25HC and PRRSV mixtures, as a result shows in incubation at room temperature 2h
Show, compared with solvent (absolute ethyl alcohol) control group, in 2.5 μM, 5 μM and 10 μM of concentration, 25HC to PRRSV virus titer without
Downward is acted on (see Fig. 2A), illustrates it to PRRSV virion without deactivation.
MARC-145 cells (Fig. 2 B) and PAMs (Fig. 2 C) are infected with PRRSV (JXwn06, MOI 0.01), after infection
Different time points handle cell with 25HC, as a result show, compared with solvent (absolute ethyl alcohol) control group, in the early stage 25HC of infection
PRRSV virus titers can be made to reduce by 1/100 to 1/10, wherein 6h handles cell with 25HC after infection, virus titer is reduced to
About 1/100, but phase 25HC effect gradually weakens after infection, and wherein 24h handles cell with 25HC after infection, 25HC can not
Reduce PRRSV virus titers (Fig. 2 B).In PAMs, 25HC has similar effect (Fig. 2 C), after this explanation PRRSV infection,
25HC remains to significantly inhibit PRRSV duplications.
2.3 25HC suppress PRRSV absorption and entrance
In order to further determine that 25HC plays a role in stage in which of PRRSV replicative cycles, 25HC is studied first is
No suppression PRRSV absorption.25HC and PRRSV (JXwn06) mixed liquor are inoculated with to the MARC-145 cells (figure of 4 DEG C of precoolings
3A) with PAMs (Fig. 3 B), 2h is made in 4 DEG C of senses, makes viruses adsorption to cell surface without entering, and is washed after virus infection with precooling PBS
Cell 3 times, detects virus titer after then adding fresh medium, 37 DEG C of culture 48h, as a result shows, with solvent (anhydrous second
Alcohol) control group compares, and when PRRSV MOI is equal to 10, on PRRSV virus titers, without influence, but in low MOI, (1 arrives 25HC
When 0.001MOI), 25HC can make PRRSV virus titers reduce by 1/100 to 1/10, and virus titer drops especially in 0.0001MOI
It is low to arrive about 1/300 (Fig. 3 A).In PAMs, compared with solvent (absolute ethyl alcohol) control group, when PRRSV MOI is equal to 0.1,
25HC on PRRSV virus titers without influence, but PRRSV MOI be 0.01 and 0.001 when, 25HC can make PRRSV virus titers
1/10 and 1/20 (Fig. 3 B) is reduced to, this explanation 25HC can suppress PRRSV absorption.
Whether research 25HC suppresses PRRSV entrance.PRRSV (JXwn06) is inoculated with to the MARC-145 cells of 4 DEG C of precoolings
2h is made in (Fig. 3 C) and PAMs (Fig. 3 D), 4 DEG C of senses, makes viruses adsorption to cell surface without entering, and precooling PBS is used after virus infection
Wash cell 3 times, then add the fresh medium containing 25HC, 37 DEG C of effect 3h abandon supernatant, cell washed with precooling PBS 3 times,
Virus titer is detected after supplementing fresh medium, 37 DEG C of culture 48h, is as a result shown, compared with solvent (absolute ethyl alcohol) control group,
25HC can make PRRSV virus titers reduce by 1/300 to 1/10, wherein, when PRRSV MOI is equal to 10,25HC can make PRRSV diseases
Malicious titre is reduced to 1/10, in 0.0001MOI, and 25HC can make PRRSV virus titers be reduced to 1/300 (Fig. 3 C).In PAMs
In, compared with solvent (absolute ethyl alcohol) control group, when PRRSV MOI is equal to 0.1,25HC on PRRSV virus titers without influence,
But when PRRSV MOI is 0.01 and 0.001,25HC can make PRRSV virus titers be reduced to 1/10 (Fig. 3 D), this explanation
25HC can significantly inhibit PRRSV entrance.
2.4 25HC do not suppress the synthesis of PRRSV genomes and the release of virion
In order to further verify synthetically produced influence that can 25HC on PRRSV genomes.By PRRSV (JXwn06, MOI
0.1) MARC-145 cells and PAMs2h are inoculated with 37 DEG C, then with (10 μM) processing cells of 25HC, and after virus infection not
Check and accept collection cell extraction RNA detection PRRSV genome contents with the time, as a result show, in MARC-145 cells (Fig. 4 A) and
In PAMs (Fig. 4 B), compared with solvent (absolute ethyl alcohol) control group, 25HC is to the relative quantity of PRRSV geneome RNAs without notable shadow
Ring, this explanation 25HC do not suppress the PRRSV assortments of genes into.
Can 25HC be studied produce influence to PRRSV release, and PRRSV (JXwn06, MOI 0.1) is inoculated with into MARC-145
24h abandons supernatant after cell, inoculation, and adds the fresh 5%DMEM containing 25HC, and the different time points after liquid is changed are collected respectively
Supernatant cell, detects virus titer.As a result show, in culture supernatant, compared with solvent (absolute ethyl alcohol) control group, 25HC
PRRSV virus titers are not made significant difference；In cell, compared with solvent (absolute ethyl alcohol) control group, 25HC is also to PRRSV diseases
Malicious titre does not make significant difference (Fig. 4 C), illustrates that releases of the 25HC to PRRSV does not make significant difference.In vitro test confirms that 25HC can pass through
Suppress PRRSV absorption and enter and significantly inhibit PRRSV duplications.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
SEQUENCE LISTING
Sequence table
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<120>A kind of medicine for suppressing the intracellular duplication of porcine reproductive and respiratory syndrome virus
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Claims (10)
1. a kind of medicine for suppressing the intracellular duplication of porcine reproductive and respiratory syndrome virus, it is characterised in that contain 25- hydroxyl courages
Sterol.
2. a kind of composition for preventing and/or treating porcine reproductive and respiratory syndrome, it is characterised in that solid containing 25- hydroxyls courage
Alcohol.
3. the pharmaceutical preparation that the medicine described in claim 1 is made with medically acceptable carrier or excipient.
4.25- hydroxy cholesterols or application of the composition in the medicine for suppressing PRRSV duplications is prepared containing it.
5.25- hydroxy cholesterols or application of the composition in the medicine that suppression PRRSV is replicated in the cell is prepared containing it.
6.25- hydroxy cholesterols or application of the composition in the medicine for suppressing PRRSV absorption host cells is prepared containing it.
7.25- hydroxy cholesterols or composition containing it suppress the application that PRRSV enters in the medicine of host cell preparing.
8. the application as described in claim 4-7 is any, described 25-HYDROXY CHOLESTEROL is made to PRRS virion without inactivation
With.
9. the application as described in claim 4-7 is any, the 25-HYDROXY CHOLESTEROL does not suppress the virus genomic conjunctions of PRRS
Into.
10. the application as described in claim 4-7 is any, the 25-HYDROXY CHOLESTEROL does not suppress the release of PRRS virion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710465206.5A CN107308168A (en) | 2017-06-19 | 2017-06-19 | A kind of medicine for suppressing the intracellular duplication of porcine reproductive and respiratory syndrome virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710465206.5A CN107308168A (en) | 2017-06-19 | 2017-06-19 | A kind of medicine for suppressing the intracellular duplication of porcine reproductive and respiratory syndrome virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107308168A true CN107308168A (en) | 2017-11-03 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609661A (en) * | 2018-12-28 | 2019-04-12 | 山东中医药大学 | A kind of combination of kidney-yang deficiency exogenous disease mouse model lung tissue qPCR reference gene and its screening technique |
| CN112471013A (en) * | 2020-10-30 | 2021-03-12 | 山东大学 | Application of 25-hydroxycholesterol in preparation of aquatic organism disease control preparation |
| CN114404439A (en) * | 2022-02-11 | 2022-04-29 | 山东农业大学 | Blocking agent for inhibiting different types of porcine reproductive and respiratory syndrome virus infection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105362278A (en) * | 2014-09-01 | 2016-03-02 | 苏州系统医学研究所 | Preparation containing 25-hydroxycholesterol and preparation method thereof and anti-virus application |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105362278A (en) * | 2014-09-01 | 2016-03-02 | 苏州系统医学研究所 | Preparation containing 25-hydroxycholesterol and preparation method thereof and anti-virus application |
Non-Patent Citations (2)
| Title |
|---|
| CHUNFENG LI, ET AL.: "25-Hydroxycholesterol Protects Host against Zika Virus Infection and Its Associated Microcephaly in a Mouse Model", 《IMMUNITY.》 * |
| 秦晓峰 等: "抵御新发、突发病毒性传染病的新思路—天然免疫机制的系统医学研究和针对宿主的广谱抗病毒药物的研发", 《中国科学：生命科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609661A (en) * | 2018-12-28 | 2019-04-12 | 山东中医药大学 | A kind of combination of kidney-yang deficiency exogenous disease mouse model lung tissue qPCR reference gene and its screening technique |
| CN112471013A (en) * | 2020-10-30 | 2021-03-12 | 山东大学 | Application of 25-hydroxycholesterol in preparation of aquatic organism disease control preparation |
| CN114404439A (en) * | 2022-02-11 | 2022-04-29 | 山东农业大学 | Blocking agent for inhibiting different types of porcine reproductive and respiratory syndrome virus infection |
| CN114404439B (en) * | 2022-02-11 | 2023-07-11 | 山东农业大学 | Blocking agent for inhibiting different types of porcine reproductive and respiratory syndrome virus infection |
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