CN110179798A - Application of the G4 ligand PhenDC3 in the drug for preparing anti-porcine reproductive and respiratory syndrome virus - Google Patents
Application of the G4 ligand PhenDC3 in the drug for preparing anti-porcine reproductive and respiratory syndrome virus Download PDFInfo
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- CN110179798A CN110179798A CN201910574825.7A CN201910574825A CN110179798A CN 110179798 A CN110179798 A CN 110179798A CN 201910574825 A CN201910574825 A CN 201910574825A CN 110179798 A CN110179798 A CN 110179798A
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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- A—HUMAN NECESSITIES
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- A61P31/12—Antivirals
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Abstract
The invention discloses application of the G4 ligand PhenDC3 in the drug for preparing anti-porcine reproductive and respiratory syndrome virus, and the application in the drug of preparation prevention and treatment porcine reproductive and respiratory syndrome.The present invention passes through Real-Time PCR experiment, Western Blot experiment and TCID50Verify influence of the PhenDC3 to PRRSV.Experiment shows that on MARC-145 cell, PhenDC3 is able to suppress PRRSV proliferation, is expected to become a kind of drug of novel prevention and treatment porcine reproductive and respiratory syndrome, have a good application prospect in terms of porcine reproductive and respiratory syndrome prevention and treatment.
Description
Technical field
The present invention relates to application of the G4 ligand PhenDC3 in the drug for preparing anti-porcine reproductive and respiratory syndrome virus,
Belong to veterinary pharmaceutical technical field.
Background technique
The advanced stage 1980s, porcine reproductive and respiratory syndrome (Porcine reproductive and
Respiratory syndrome, PRRS), also known as pig blue-ear disease is most reported early in the U.S..The disease Clinical symptoms is farrowing sow
Miscarriage produces stillborn foetus, the mummification of fetus and weak son and respiratory disease, brings to worldwide pig breeding industry serious
Economic loss.
PRRS virus (PRRSV) in China's prevalence, represents strain CH-1a since the nineteen ninety-five, is american type PRRSV-2.
Since 2006, highly pathogenic PRRSV (8 system of HP-PRRSV, Lineage) pandemic at home is first-born more than 20,000,000
Pig is infected, and representing strain includes JXA1, HUN4 etc..2010, it was PRRSV strain that Guangdong Province, which is separated to one plant of Lineage 3,
Research shows that infection is mainly distributed on China southeast, pathogenecity is weaker, represents strain as QYYZ and GM2.2013 with
Come, Lineage 1 is that PRRSV starts in Chinese pandemic, and clinical recall rate rises year by year.Because with U.S. NADC30 poison
Strain homology is high, referred to as NADC30-like strain.Wide due to being related to region, pathogenicity is strong, it is prone to recombinate, cause people
Extensive concern.
Belong to shell type virales, Arteriviridae in PRRSV classification.PRRSV is the single-stranded positive RNA disease for having cyst membrane
Poison, full-length genome about 15kb.The reproduction process of PRRSV is template first with geneome RNA (gRNA), synthesizes strand RNA
Intermediate.Then, using minus-strand RNA intermediate as template, synthesize normal chain filial generation geneome RNA and a series of subgenomes
RNA(sgmRNA).These subgenomic RNAs (sgmRNA) contain ORF1a, ORF1b, ORF2a, ORF2b, ORF3-ORF7 etc.
Open reading frame.The polyprotein of ORF1a and ORF1b coding can be with self cleavage for multiple non-structural protein (Nonstructural
Protein, Nsp), including Nsp1 α, Nsp1 β, Nsp2-12 etc..ORF2a, ORF2b, ORF3-ORF7 encode multiple structural proteins,
Such as GP2a, GP2b, GP3, GP4, GP5a, GP5, M albumen and N protein.Existing PRRSV vaccine and drug cause a disease mainly for height
Property strain (HP-PRRSV), and specific medicament there is no for NADC30-like strain currently popular.Also, due to PRRSV core
Acid sequence is prone to make a variation, and conventional medicine is difficult to form effective protection.
Tetra- serobila of G (G4) is a kind of spy that the nucleic acid rich in guanine base (guanine, G) is formed under certain condition
Different secondary structure.In the presence of G4 ligand, in chain or the G of interchain is by Hoogsteen key, can form stable G4 knot
Structure.Studies have shown that G4 structure is related to the regulation of telomere function and cancer.G4 ligand-PhenDC3 passes through the area for combining and being rich in G
Domain inhibits tumour cell telomerase activation, is expected to become novel anticancer drug.Function of the PhenDC3 in PRRSV proliferation at present
Can be unclear, also have no that pertinent literature discloses.
Summary of the invention
The status that the purpose of the present invention is change for PRRSV current popular strain and antiviral drugs lacks, proposes one
Kind novel substance-application of the G4 ligand PhenDC3 in the drug for preparing anti-porcine reproductive and respiratory syndrome virus, it is described
PhenDC3 has good antivirus action to PRRSV NADC30-like strain, before having application well in terms of the PRRS prevention and treatment
Scape.
To achieve the goals above, the technical scheme adopted by the invention is that:
Application of the G4 ligand PhenDC3 in the drug for preparing anti-porcine reproductive and respiratory syndrome virus.
The structural formula of G4 ligand PhenDC3 is as follows:
The porcine reproductive and respiratory syndrome virus is NADC30-like strain.
A kind of drug of anti-porcine reproductive and respiratory syndrome virus, the G4 ligand PhenDC3 comprising effective dose.
The effective dose of G4 ligand PhenDC3 is 5-10 μM.
The drug is spray, injection, tablet, capsule or granule.
Application of the G4 ligand PhenDC3 in the drug of preparation prevention and treatment porcine reproductive and respiratory syndrome.
A kind of drug for preventing and treating porcine reproductive and respiratory syndrome, the G4 ligand PhenDC3 comprising effective dose.
The effective dose of G4 ligand PhenDC3 is 5-10 μM.
The drug is spray, injection, tablet, capsule or granule.
The invention has the advantages that:
The invention discloses G4 ligand-PhenDC3 in the medicine for preparing anti-porcine reproductive and respiratory syndrome virus (PRRSV)
Application in object, and the application in the drug of preparation prevention and treatment porcine reproductive and respiratory syndrome.The present invention passes through Real-Time
PCR experiment, Western Blot experiment and TCID50Verify influence of the PhenDC3 to PRRSV.Experiment shows thin in MARC-145
On born of the same parents, PhenDC3 is able to suppress PRRSV proliferation, is expected to become a kind of medicine of novel prevention and treatment porcine reproductive and respiratory syndrome
Object has a good application prospect in terms of porcine reproductive and respiratory syndrome prevention and treatment.
Detailed description of the invention
Fig. 1 is PhenDC3 cell toxicity test result.
Fig. 2 is the inhibitory effect that PhenDC3 is proliferated PRRSV in rna level on MARC-145 cell.
Fig. 3 is the inhibitory effect that PhenDC3 synthesizes PRRSV N protein on MARC-145 cell.
Fig. 4 is inhibitory effect of the PhenDC3 to PRRSV titre on MARC-145 cell.
Specific embodiment
A specific embodiment of the invention is further described with reference to embodiments, but embodiment is not to this hair
It is bright to limit in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus for this field conventional reagent,
Method and apparatus.Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The statistical analysis of following embodiment of the present invention: all tests at least 3 times independent to repeat, as a result using average value and
Standard error indicates, uses one-way analysis of variance and t check analysis.All statistical analysis are all made of using P < 0.01 * * as having
The test stone of extremely significant statistical difference, analysis software are GraphPad Prism 5.
Embodiment 1:PhenDC3 cell toxicity test
Every hole 1 × 104A MARC-145 cell inoculation to containing 10% (v/v) fetal calf serum DMEM culture medium 96 holes
In plate.After cell is adherent, culture medium is discarded, the maintaining liquid containing 2.5 μM, 5 μM, 10 μM and 20 μM of PhenDC3 is changed to and (contains
The DMEM culture medium of 2% (v/v) fetal calf serum), every 200 μ l of hole, every kind of concentration repeats 3 holes.Setting simultaneously, which adds, contains 2% (v/v)
The DMSO cell controls group (i.e. PhenDC3 is 0 μM) of the maintaining liquid of DMSO.37 DEG C, 5%CO2Continue after cultivating 3d in incubator,
5mg/ml MTT (3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide) solution is added in every hole and (is dissolved in PBS
In) 20 μ l, continue in incubator to terminate after being incubated for 4h.It inhales and abandons culture supernatant in hole, 150 μ l DMSO are added in every hole, anti-to MTT
The blue crystal answered sufficiently dissolves.570nm wavelength is selected, the light absorption value in each hole is measured on enzyme linked immunological monitor, is recorded
As a result.With the cell activity of DMSO cell controls group for 100%, in the read-around ratio of the PhenDC3 processing group cell of various concentration
The reading of DMSO cell controls group is versus cell activity.
As a result there is no toxicity to MARC-145 cell, carefully when PhenDC3 concentration is 20 μM and is following as shown in Figure 1:
Cytoactive reaches 95% or more.
Embodiment 2: on MARC-145 cell, inhibitory effect that PhenDC3 is proliferated PRRSV in rna level
MARC-145 cell is cultivated on six orifice plates of the DMEM culture medium containing 10% (v/v) fetal calf serum.It discards afterwards for 24 hours
The PRRSV (NADC30-like strain) of MOI=0.1 is connect poison by culture medium.It is cultivated with the DMEM containing 2% (v/v) fetal calf serum
Base (every 200 μ l of hole), in 5%CO22h is cultivated under the conditions of 37 DEG C of constant incubator.Culture medium is discarded, is cleaned twice with PBS, point
Not be 0 μM containing concentration, 2.5 μM, 5 μM, 10 μM of PhenDC3, DMEM culture medium (every 200 μ of hole of 2% (v/v) fetal calf serum
L), in 5%CO236h is cultivated under the conditions of 37 DEG C of constant incubator.In -80 DEG C and 4 DEG C six orifice plate of multigelation 3 times, fill cell
Division solution, collects each hole lysate, extracts total serum IgE, and carry out reverse transcription immediately.Reverse transcription reagent box (Transcriptor
First Strand cDNA Synthesis Kit) it is purchased from Roche company.
The first step, reaction system: 1.0 μ L oligo (dT)18Primer, 1 μ g total serum IgE, 12.0 Hs of the μ L without RNA enzyme2O.Instead
Answer condition are as follows: 65 DEG C of 10min.
Second step, reaction system: 4.0 μ L 5 × RT Buffer, 2.0 μ L Deoxynucleotide Mix, 0.5 μ L
Protector RNase Inhibitor,0.5μL Transcriptor Reverse Transcriptase.By above-mentioned system
It is mixed with first step reaction system.Reaction condition are as follows: 55 DEG C of 30min, 85 DEG C of 5min.
For the cDNA obtained using above-mentioned reverse transcription as template, GAPDH is reference gene, using primer, carries out Real-Time
PCR amplification detects the copy number of the N gene of PRRSV.Real-time PCR reagent (SYBR qPCR Mix) is purchased from ABI company.
Reaction system: 5.0 μ 2 × SYBR of L qPCR Mix, 4.0 Hs of the μ L without RNA enzyme2O, 0.25 μ L upstream primer (10 μM), 0.25 μ L
Downstream primer (10 μM), 0.5 μ L cDNA.Reaction condition are as follows: 95 DEG C of 10min carry out initial denaturation reaction, follow subsequently into 40
Ring.Circular response is 94 DEG C of 10s, 56 DEG C of 15s, 72 DEG C of 15s.
N gene upstream and downstream primer sequence:
N-F:5 '-CAGTCCAGAGGCAAGGGA-3 ' (SEQ ID NO.1)
N-R:5 '-CGGCAAACTAAACTCCACA-3 ' (SEQ ID NO.2)
GAPDH gene upstream and downstream primer sequence:
GAPDH-F:5 '-TCATGACCACAGTCCATGCC-3 ' (SEQ ID NO.3)
GAPDH-R:5 '-GGATGACCTTGCCCACAGCC-3 ' (SEQ ID NO.4)
Test result is as shown in Fig. 2, show that PhenDC3 of the invention in 5-10 μM of concentration range, exists to PRRSV
The synthesis of RNA significantly inhibits effect on MARC-145 cell.
Embodiment 3: on MARC-145 cell, inhibitory effect that PhenDC3 synthesizes PRRSV N protein
MARC-145 cell is cultivated on six orifice plates of the DMEM culture medium containing 10% (v/v) fetal calf serum.It discards afterwards for 24 hours
The PRRSV (NADC30-like strain) of MOI=0.1 is connect poison by culture medium.It is cultivated with the DMEM containing 2% (v/v) fetal calf serum
Base (every 200 μ l of hole), in 5%CO22h is cultivated under the conditions of 37 DEG C of constant incubator.Culture medium is discarded, is cleaned twice with PBS, point
Not be 0 μM containing concentration, 2.5 μM, 5 μM, 10 μM of PhenDC3, DMEM culture medium (every 200 μ of hole of 2% (v/v) fetal calf serum
L), in 5%CO236h is cultivated under the conditions of 37 DEG C of constant incubator.Culture medium in 6 orifice plates is discarded, is cleaned twice with the PBS of pre-cooling,
120 μ l cell pyrolysis liquids (protease inhibitors containing cocktail) are added in every hole, shake up, in standing 5min on ice, use cell scraper
Scraping cells carry out Western Blot experiment.Protein concentration in sample is measured, SDS-PAGE is used for.
SDS-PAGE electrophoresis: applied sample amount, 20 μ g samples on every hole are determined according to protein concentration.Power on, voltage is transferred to
Voltage is transferred to 120V when bromophenol blue indicator goes to separation gel by 80V, and bromophenol blue indicators is waited to go to separation gel bottom (about
Need 2h) when, terminate electrophoresis.
Transferring film: the gel after taking SDS-PAGE electrophoresis directly uses Bio-RAD company Mini Trans-Blot
Albumen electrification is transferred on pvdf membrane by Electrophoretic Transfer Cell transfer device.Concrete operations are as follows:
A small amount of transferring film buffer is added in one pallet, filter paper and sponge are soaked in wherein.After pvdf membrane impregnates 2min in methyl alcohol
Move into transferring film buffer.Sponge, filter paper are successively spread on a glass, drive bubble away with glass bar, the glue after electrophoresis is put down gently
On filter paper, glass bar drives bubble away, sticks pvdf membrane, is then covered with filter paper and sponge, and glass bar drives bubble away.By its global transfer
Onto transferring film folder.Transferring film 3-4h under 40V voltage puts ice bag in its surrounding.
Pvdf membrane is pressed from both sides out with tweezers and is laid in the confining liquid containing 5% (w/v) BSA, sets and is incubated for 2h on shaking table.TBST is washed
3 times, each 5min.Flushing liquor is outwelled, primary antibody solution (PRRSV N protein monoclonal antibody 1:500 dilution, internal reference β-is added
Actin 1:2000 dilution), 4h is incubated on shaking table.TBST is washed 3 times, each 5min.Flushing liquor is outwelled, two corresponding anti-solution is added
(the sheep anti mouse secondary antibody 1:3000 dilution of FITC label), is incubated for 2h.Pvdf membrane is put into chemiluminescent substrate mixed liquor and is carried out
It is protected from light colour developing, is imaged with chemiluminescence imaging system.
Test result is as shown in figure 3, show that PhenDC3 of the invention in 2.5-10 μM of concentration range, exists to PRRSV
The synthesis of N protein significantly inhibits effect on MARC-145 cell, and dose dependent is presented.Concentration of the PhenDC3 at 10 μM
When, it can not detect the synthesis of N protein.
Embodiment 4: on MARC-145 cell, inhibitory effect of the PhenDC3 to PRRSV titre
MARC-145 cell is cultivated on six orifice plates of the DMEM culture medium containing 10% (v/v) fetal calf serum.It discards afterwards for 24 hours
The PRRSV (NADC30-like strain) of MOI=0.1 is connect poison by culture medium.It is cultivated with the DMEM containing 2% (v/v) fetal calf serum
Base (every 200 μ l of hole), in 5%CO22h is cultivated under the conditions of 37 DEG C of constant incubator.Culture medium is discarded, is cleaned twice with PBS, point
Not be 0 μM containing concentration, 2.5 μM, 5 μM, 10 μM of PhenDC3, DMEM culture medium (every 200 μ of hole of 2% (v/v) fetal calf serum
L), in 5%CO236h is cultivated under the conditions of 37 DEG C of constant incubator.In -80 DEG C and 4 DEG C six orifice plate of multigelation 3 times, fill cell
Division solution, collects each hole lysate, with TCID50Method detects PRRSV titre.
TCID50The step of detection method, is: sample to be tested DMEM culture medium is made 10 times of series in 1.5mL EP pipe
Dilution, i.e., 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9、10-10, to be replaced when transfer liquid in dilution
Pipette tips.By the poison disease vaccination of each dilution into 96 hole cell micro culture plate 1-10 column, 8 hole of each column, every hole inoculation
100μL.11-12 column plus the DMEM culture medium of equivalent compare.MARC-145 cell confluent monolayers in 96 orifice plates.By 96 holes
Cell micro culture plate is placed in 37 DEG C, 5%CO272h is cultivated in incubator, record cell generates lesion hole count.By Reed-
Muench method calculates virus TCID to be measured50Value.
Test result is as shown in figure 4, show that PhenDC3 of the invention in 5-10 μM of concentration range, exists to PRRSV
Viral titer on MARC-145 cell significantly inhibits effect.
Sequence table
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<120>application of the G4 ligand PhenDC3 in the drug for preparing anti-porcine reproductive and respiratory syndrome virus
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Claims (10)
- Application of the 1.G4 ligand PhenDC3 in the drug for preparing anti-porcine reproductive and respiratory syndrome virus.
- 2. application according to claim 1, which is characterized in that the structural formula of G4 ligand PhenDC3 is as follows:
- 3. application according to claim 1, which is characterized in that the porcine reproductive and respiratory syndrome virus is NADC30- Like strain.
- 4. a kind of drug of anti-porcine reproductive and respiratory syndrome virus, which is characterized in that the G4 ligand comprising effective dose PhenDC3。
- 5. drug according to claim 4, which is characterized in that the effective dose of G4 ligand PhenDC3 is 5-10 μM.
- 6. drug according to claim 4, which is characterized in that the drug is spray, injection, tablet, capsule Or granule.
- Application of the 7.G4 ligand PhenDC3 in the drug of preparation prevention and treatment porcine reproductive and respiratory syndrome.
- 8. a kind of drug for preventing and treating porcine reproductive and respiratory syndrome, which is characterized in that the G4 ligand comprising effective dose PhenDC3。
- 9. drug according to claim 8, which is characterized in that the effective dose of G4 ligand PhenDC3 is 5-10 μM.
- 10. drug according to claim 8, which is characterized in that the drug is spray, injection, tablet, capsule Or granule.
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