CN109679952A - A kind of pig source miR-c89 of anti-PRRSV infection and its application - Google Patents
A kind of pig source miR-c89 of anti-PRRSV infection and its application Download PDFInfo
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Abstract
The invention discloses a kind of pig source miR-c89 of anti-PRRSV infection, highly pathogenic and low pathogenicity PRRSV duplication and proliferation can be significantly inhibited from the miR-c89 of pig coding by being demonstrated for the first time by the method for RT-qPCR, TCID50 and western blot, the micoRNA is expected to be developed into a kind of development of novel drug and the gene modification pig for anti-pig blue-ear disease for preventing and treating pig blue-ear disease, lays the foundation for the prevention and control of PRRSV.
Description
Technical field
The present invention relates to field of biotechnology, the pig source miR-c89 of more particularly to a kind of anti-PRRSV infection and
It is applied.
Background technique
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,
It PRRS is) viral (Porcine reproductive and respiratory syndrome virus, PRRSV) by PRRS
It is caused using the symptoms such as sow breeding difficulty and piglet respiratory tract be impaired as the viral infectious of main feature.The disease is in 1987
Year is broken out in the U.S. for the first time, then propagates and popular, has brought tremendous economic losses in the world to global pig breeding industry.
South China pig farm in 2006 has been broken out using high fever, high incidence, high mortality as " porcine hyperthermia " of main feature, and causing should
The cause of disease of disease is finally proved to be a kind of PRRSV of variation, is named as highly pathogenic PRRSV (Highly pathogenic
PRRSV, HP-PRRSV).Compared with classical strains, HP-PRRSV can infect the pig of all ages and classes, different phase and different sexes
Group, and show high incidence and the death rate.The prevalence of HP-PRRSV brings heavy strike to China's pig breeding industry again.By
Have antigenic variability, thermophilic phagocytic, antibody-dependent enhancement (ADE) and persistent infection etc. special in PRRS virus
Sign, existing vaccine is limited to the protective effect of the disease, and there is presently no the specific medicaments of anti-PRRS virus.
MicroRNA is a kind of by the length of endogenous gene be about 22 nucleotide non-coding single strand RNA molecule,
It plays extremely important post-transcriptional control in the interaction of virus and host and acts on.On the one hand, virus infection can
Lead to the change of host-encoded microRNA expression, the latter is raw to virus by targeting virus or the gene of itself
All too many levels of life process are regulated and controled.On the other hand, many viruses can equally encode microRNA and adjust host and virus
The expression of autogene plays a significant role during virus infection host cell.
In terms of virus infection, microRNA is applied to clinical test as the inhibitor of hepatitis c virus infection.And
There is research to confirm during PRRSV infection host cell, host-encoded microRNA plays the part of weight for the infection of PRRSV
Want regulating and controlling effect.Therefore it provides a kind of pig source miR-c89 with anti-PRRSV infection, is used to prepare prevention or treatment pig indigo plant ear
The problem of drug or preparation of disease are those skilled in the art's urgent need to resolve.
Summary of the invention
In view of this, the present invention provides a kind of pig source miR-c89 with anti-PRRSV infection, can be used for preparing prevention
Or the drug or preparation for the treatment of pig blue-ear disease, and the gene modification pig of anti-pig blue-ear disease is developed, it realizes to the effective of pig blue-ear disease
Prevention and control.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of pig source miR-c89 sequence of anti-PRRSV infection, the miR-c89 nucleotide sequence coded RNA sequence is such as
Under:
GUACAGUACUGUGAUAACUGA；SEQ ID NO.1.
Further, application of the pig source miR-c89 in the drug or preparation of preparation prevention or treatment pig blue-ear disease.
Further, the miR-c89 sequence transfection PAMs cell can inhibit duplication and increasing of the PRRSV in PAMs cell
It grows.
Further, the precursor RNA of the pig source miR-c89 sequence of a kind of anti-PRRSV infection, the miR-c89 nucleotides sequence
The precursor RNA sequence for arranging coding is as follows:
GGUUAUCAUGGUACCGAUGCUGUAUAUCUGAAAGGUACAGUACUGUGAUAACUGA；SEQ ID NO.2.
Further, the precursor RNA of the miRNA of the pig source miR-c89 sequential coding is in preparation prevention or treatment pig indigo plant ear
Application in the drug or preparation of disease.
Further, the precursor RNA transfection PAMs cell of the miRNA of the pig source miR-c89 sequential coding can inhibit PRRSV
Duplication and proliferation in PAMs cell.
Further, the precursor RNA of the miRNA of the pig source miR-c89 sequential coding is repaired in the gene for developing anti-blue otopathy
Adorn the application in pig.
It can be seen via above technical scheme that compared with prior art, the present disclosure provides a kind of anti-PRRSV infections
Pig source miR-c89, by the method for RT-qPCR, TCID50 and western blot demonstrate for the first time from pig coding
MiR-c89 can significantly inhibit highly pathogenic and duplication and proliferation of low pathogenicity PRRSV, which is expected to be developed into
A kind of development of the novel drug and the gene modification pig for anti-pig blue-ear disease for preventing and treating pig blue-ear disease, is the prevention and control of PRRSV
It lays the foundation.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 attached drawing is that the present invention transfects miR-c89 analogies postoperative infection HP-PRRSV GD-HD strain in PAMs cell,
The relative expression quantity of PRRSV ORF7；
Fig. 2 attached drawing is that the present invention transfects miR-c89 analogies postoperative infection HP-PRRSV GD-HD strain in PAMs cell
When, the western blot testing result of PRRSV N protein；
Fig. 3 attached drawing is that the present invention transfects miR-c89 analogies postoperative infection HP-PRRSV GD-HD strain in PAMs cell,
PRRSV genome copy numbers in PAMs cell culture supernatant；
When Fig. 4 attached drawing is that the present invention transfects miR-c89 analogies postoperative infection GD-HD strain in PAMs cell, cell training
Support the virus titer in supernatant；
Wherein, in Fig. 1-Fig. 4, miR-c89 analogies miR-c89 analogies: are transfected in cell；Compare analogies:
Unrelated miRNA analogies are transfected in cell；Normal cell controls: transfection reagent is only added in cell and is added without analogies；It is empty
White control: transfection reagent is added without in cell and is also added without analogies；
When Fig. 5 attached drawing is that the present invention transfects miR-c89 analogies postoperative infection N-PRRSV CH1a strain in PAMs cell,
The relative expression quantity of PRRSV ORF7；
Fig. 6 attached drawing is that the present invention transfects miR-c89 analogies postoperative infection N-PRRSV CH1a strain in PAMs cell,
PRRSV genome copy numbers in PAMs cell culture supernatant；
When Fig. 7 attached drawing is that the present invention transfects miR-c89 analogies postoperative infection N-PRRSV CH1a strain in PAMs cell,
Virus titer in cell culture supernatant；
Wherein, in Fig. 5-Fig. 7, the experimental group of miR-c89 analogies miR-c89 analogies: is transfected in cell；Compare mould
Quasi- object: the experimental group of unrelated miRNA analogies is transfected in cell；Normal cell controls: transfection reagent is only added in cell
It is added without the experimental group of analogies；Blank control: the experimental group that transfection reagent is also added without analogies is added without in cell；
Fig. 8 attached drawing is to transfect miR-c89 analogies after the present invention infects HP-PRRSV GD-HD strain in PAMs cell,
The relative expression quantity of PRRSV ORF7；
Fig. 9 attached drawing is to transfect miR-c89 analogies after the present invention infects HP-PRRSV GD-HD strain in PAMs cell,
PRRSV genome copy numbers in PAMs cell culture supernatant；
Figure 10 attached drawing is to transfect miR-c89 analogies, cell culture after the present invention infects GD-HD strain in PAMs cell
Virus titer in supernatant；
Wherein, in Fig. 8-Figure 10, the experimental group of miR-c89 analogies miR-c89 analogies: is transfected in cell；Control
Analogies: the experimental group of unrelated miRNA analogies is transfected in cell；Normal cell controls: transfection examination is only added in cell
Agent is added without the experimental group of analogies；Blank control: the experimental group that transfection reagent is also added without analogies is added without in cell；
Figure 11 attached drawing is to transfect miR-c89 analogies after the present invention infects N-PRRSV CH1a strain in PAMs cell,
The relative expression quantity of PRRSV ORF7；
Figure 12 attached drawing is to transfect miR-c89 analogies after the present invention infects N-PRRSV CH1a strain in PAMs cell,
PRRSV genome copy numbers in PAMs cell culture supernatant；
Figure 13 attached drawing is to transfect miR-c89 analogies after the present invention infects N-PRRSV CH1a strain in PAMs cell,
Virus titer in cell culture supernatant；
Wherein, in Figure 11-Figure 13, the experimental group of miR-c89 analogies miR-c89 analogies: is transfected in cell；Control
Analogies: the experimental group of unrelated miRNA analogies is transfected in cell；Normal cell controls: transfection examination is only added in cell
Agent is added without the experimental group of analogies；Blank control: the experimental group that transfection reagent is also added without analogies is added without in cell；
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
DMEM culture medium, Opti-MEM culture medium and RPMI-1640 culture medium are purchased from Life Tech company；Trypsase
BI company is purchased from fetal calf serum；MicroRNA analogies are purchased from Guangzhou Rui Bo Biotechnology Co., Ltd；Primer is by Shanghai
The synthesis of Invitrogen company；FastStart Universal SYBR Green Master and siRNA transfection reagent is purchased from
Roche company；HS enzyme, RNA extract reagent (RNAiso Plus), reverse transcription reagent box
(PrimeScriptTM RT Reagent Kit) is purchased from Takara company；Trans5 α competent cell, DNA purification kit
(Easy Pure Quick Gel Extraction kit) is purchased from Beijing Quan Shijin biotech company；PsiCheck2 carrier
And Dual-Luciferase Dual-luciferase reportor systerm is purchased from Promega company；PAMs cell is that (pig alveolar macrophage is thin
Born of the same parents) it is located away from the lung tissue of 6 weeks piglets；MARC-145 cell (the derived cell system of African green monkey kidney cell MA-104) is high
Pathogenic PRRSV strain GD-HD (GenBank ID:KP793736.1) and low pathogenicity PRRSV strain CH1a (GenBank
ID:AY032626.1 it) is saved by Xibei Univ. of Agricultural & Forest Science & Technology's animal doctor's Pathogen Biology laboratory.
50nM miR-c89 analogies, miR- when then infecting highly pathogenic HP-PRRSV are first transfected in embodiment 1PAMs
The influence that c89 replicates HP-PRRSV
(1) transfection of miR-c89 analogies and cell connect poison:
1) bed board: separating porcine alveolar macrophage from the piglet lung tissue of 6 week old, and carries out cell count and paving
Plate, in 37 DEG C of 5%CO2It is cultivated in incubator；
2) transfect: transfection reagent is the X-tremeGENE siRNA Transfection Reagent of Roche Holding Ag, is pressed
It is transfected according to the operating procedure of transfection reagent.Transfection cocktail (analogies and siRNA are prepared after bed board 12h
Transfection reagent is dissolved in Opti-MEM respectively, then the two is mixed) incubation at room temperature 20min.By 24 orifice plates from
It is taken out in incubator, changes Opti-MEM into after being cleaned with PBS, mixture is added dropwise and rocks mixing, is placed in cell incubator
Culture；
3) connect poison: transfection is followed by poison for 24 hours, is inoculated with HP-PRRSV GD-HD strain with 0.1 MOI, thin according to formula PFU=
Born of the same parents' number × MOI=0.7 × TCID50 calculates required virus liquid；24 orifice plates are taken out, after being cleaned with PBS, it is dilute that virus is added
Liquid is released, connect virus liquid is discarded after 37 DEG C of culture 1h, is changed to 1640 culture mediums of serum content 3%；
4) it receives sample: 0h, 12h after infection, collecting cell sample and cell culture supernatant, -80 DEG C of preservations for 24 hours；Cell
Sample is for detecting the expression of the relative expression levels of PRRSV ORF7 gene and N protein in cell, in cell culture
Clear liquid is discharged into virus genomic copy number and virus titer in culture solution supernatant for detecting.
(2) RT-qPCR detects PRRSV ORF7 mRNA relative expression levels:
1) RNA and reverse transcription extracted in cell is cDNA；
Total serum IgE after extracting PRRSV infection PAMs cell using Takara company RNAiso Plus:
Step are as follows: after cell adds the RNAiso Plus of TAKARA company, sufficiently crack；It is sequentially added according to operating instruction
Chloroform, isopropanol, RNA is sunken to tube bottom after centrifugation；75% ethyl alcohol cleaning RNA is added, after drying to be precipitated, is added suitable
RNase-free water is dissolved；
RNA concentration, purity and integrality are measured, RNA concentration and purity, OD are measured on nucleic acid-protein analyzer260/
OD280Ratio between 1.8-2.0, concentration be greater than 2 μ g/ μ l；Agarose gel electrophoresis is carried out simultaneously, uses gel imaging system
5S rRNA, the 18S rRNA and 28SrRNA band of detected RNA are observed, shows three band complete displays.
Using Takara company reverse transcription reagent box (PrimeScriptTM RT ReagentKit) for extracted
RNA carries out reverse transcription.
Reverse transcription reaction system: for 5 × PrimeScript Buffer, 2 μ l；RT Enzyme
MixI, 0.5 μ l；Oligo dT Primer (50 μM), 0.5 μ l；Random Primer (100 μM), 0.5 μ l；Total RNA,
500ng；RNase Free ddH2O supplies 10 μ l.
The reaction condition of reverse transcription are as follows: 37 DEG C, 20min；85 DEG C, 5s；4℃.
2) qPCR detection is carried out, the mrna expression amount of PRRSV ORF7 and internal reference HPRT-1 gene in each sample are detected,
PCR reaction system (10 μ l): 2 × SYBR Green Mix, 5.0 μ l, ddH22.0 μ l of O, upstream primer (12 μM) 0.25 μ l, under
Swim primer (12 μM) 0.25 μ l, 2.5 μ l of template cDNA.
PCR response procedures are as follows: 95 DEG C of 10min；95℃15s；60℃30s；95℃15s；40 circulations.
WithThe StepOne Software v2.3 software of Real-Time PCR System instrument into
The analysis of row result.
QPCR primer are as follows:
PRRSV-ORF7-F:AGATCATCGCCCAACAAAAC；SEQ ID NO.3；
PRRSV-ORF7-R:GACACAATTGCCGCTCACTA；SEQ ID NO.4；
HPRT1-F:TGGAAAGAATGTCTTGATTGTTGAAG；SEQ ID NO.5；
HPRT1-R:ATCTTTGGATTATGCTGCTTGACC；SEQ ID NO.6.
As a result as shown in Figure 1, transfecting miR-c89 analogies postoperative infection HP-PRRSV GD-HD strain in PAMs cell,
In 12hpi and pi for 24 hours, the relative expression quantity of PRRSV ORF7 has dropped about 97.8% compared with compareing analogies group.
(3) western blot detects the expression of PRRSV N protein:
1) preparation of samples: being centrifuged 10min for the 500 × g of cell sample collected, abandons after supernatant and cell is resuspended with PBS (K+)
Precipitating, 500 × g discard supernatant after being centrifuged 10min；With the NP40 lysate lytic cell containing protease inhibitors, fill on ice
Division solution 30min；4 DEG C, 12,000 × g is centrifuged 10min, supernatant is transferred in new centrifuge tube and is marked；According to north
Protein concentration in the BCA protein quantification kit specification measurement sample of Jing Bomaide gene technology Co., Ltd；Use NP40
5 × SDS-PAGE loading buffer is added after unifying the total protein concentration of each sample in lysate, and 100 DEG C are boiled 5min；It will
The protein sample handled well carries out sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
2) SDS-PAGE: assembling after offset plate according to Bio-Rad specification and is fixed on on glue frame, prepares 12% first
Separation gel (10ml): 3.8ml ddH is sequentially added in matching glue Special conical flask2O, 30% acrylamide of 3.4ml, 2.6ml
1.5M Tris-HCl (pH 8.8), 0.1ml 10%SDS, 0.1ml 10% ammonium persulfate, 0.004ml TEMED.It is light after adding
Light shaking, which mixes, prevents bubble, and the separation gel of mixing is quickly poured into assembled offset plate layer glass plate stitch gap, is added
After to 2/3rds of offset plate height, one layer of ultrapure water is gently covered with 200 μ l pipettors on the upper layer of separation gel, room temperature is quiet
Set 30-60min.After gelling to be separated is solid, after separation gel upper layer ultrapure water is poured out and gently blotted with blotting paper, prepare immediately
5% concentration glue (4ml): ddH2O 2.8ml, 30% acrylamide 0.66ml, 1.0M Tris-HCl (PH 6.8) 0.5ml, 10%
SDS 0.04ml, 10% ammonium persulfate 0.04ml, TEMED 0.004ml.It is once added in the conical flask cleaned up above-mentioned
Component fills offset plate gap after mixing immediately, and corresponding comb is immediately inserted into after filling, is placed at room temperature for 30-60min.
After glue prepares, offset plate is removed on glue frame from matching and is inserted into electrophoresis tank according to Bio-Rad specification, by gel
In comb gently extract vertically, in well be added same volume the sample handled well and albumen Marker.To electricity
Appropriate electrophoresis liquid is added in swimming slot, powers on and is adjusted to constant pressure 200V and start electrophoresis, until bromophenol blue is migrated to separation gel bottom
Portion stops electrophoresis.
3) transferring film: after the completion of SDS-PAGE, offset plate being taken out, careful separation layer glass plate, is taken out gel and is cut off dense
Contracting glue.Remaining gel, the pvdf membrane (30s is impregnated in methanol) of activation, two pieces of filter paper and sponge are immersed in transferring film liquid；It beats
Open transferring film clip, by cathode to anode successively stack sponge, filter paper, gel, pvdf membrane, filter paper, sponge (especially attention gel with
Cannot have bubble between sponge), clip is buckled well rapidly after putting well, gel is avoided to move with pvdf membrane；It will after assembling
Clip is inserted into transferring film slot and supplements appropriate transferring film buffer, and transferring film slot is placed in ice water, in case a large amount of heat production of During migration cause
Protein degradation；Power on and is adjusted to constant pressure 100V transferring film 1-2h.
4) it closes and is incubated for antibody: closing, pvdf membrane taking-up is immersed in confining liquid after transferring film, 37 DEG C of closings
1h；Then pvdf membrane is immersed in corresponding primary antibody dilution, 2h or 4 DEG C of overnight incubation is incubated at room temperature on incubation shaking table；One
Pvdf membrane is washed in PBST 3 times after anti-incubation, each 10min；After washing film, by pvdf membrane according to the big cabinet of albumen
It opens, is infiltrated in the secondary antibody diluent of corresponding HRP label respectively, be incubated at room temperature 1h；After secondary antibody is incubated for, PBST washes film 3
It is secondary, each 10min；
5) it develops the color: after the colour developing of ECL chemical luminescence reagent kit, pvdf membrane being put into chemiluminescence colour developing imaging system, is clapped
It takes the photograph and saves picture.
As a result as shown in Fig. 2, when transfecting miR-c89 analogies postoperative infection HP-PRRSVGD-HD strain in PAMs cell,
Western blot testing result is consistent with qPCR testing result, all several before for 24 hours after infection after transfection miR-c89 analogies
It can't detect the expression of PRRSV N protein.
(4) PRRSV genome copy numbers in RT-qPCR detection assay Supernatant samples:
1) cell culture supernatant of the 400ul collected in step (1) is mixed with 400ul RNAiso Plus laggard
Row cracking is extracted RNA and is dissolved in the RNase-free water of 15 μ l；
2) reverse transcription reaction system is configured: for 5 × PrimeScript Buffer, 2 μ l；RT
Enzyme MixI, 0.5 μ l；Oligo dT Primer (50 μM), 0.5 μ l；Random Primer (100 μM), 0.5 μ l；
Total RNA, 6.5 μ l；The reaction condition of reverse transcription are as follows: 37 DEG C, 20min；85 DEG C, 5s；4℃；
3) it prepares positive criteria product (plasmid standard containing PRRSV ORF7 genetic fragment) and calculates in Supernatant samples
PRRSV genome copy numbers: the complete region CDS (372bp) of PRRSV ORF7 is cloned into pMD-18T carrier, conversion is extremely
Identification is sequenced after extracting plasmid in trans5 α competence.Measurement plasmid DNA concentration is named as the standard items of absolute quantitation
PMD-18T-ORF7 (initial concentration is 5.5ng/ μ l) carries out the gradient dilution with 10 for multiple, according to formula: copy number
(copies)=(quality/molecular weight) × 6.0 × 1023, calculate the copy number of different dilution standard items, form RT-qPCR's
Standard curve is detected with PRRSV-ORF7 RT-qPCR primer, is analyzed data with Real time PCR analyzer, is calculated
PRRSV genome copy numbers in every milliliter of cell culture supernatant out.
As a result as shown in figure 3, transfecting miR-c89 analogies postoperative infection HP-PRRSV GD-HD strain in PAMs cell
When 12hpi and for 24 hours pi, under PRRSV genome copy numbers are compared with compareing analogies group respectively in PAMs cell culture supernatant
94.3% and 96.5% have dropped.
(5) TCID50 measures the titre of PRRS virus in Supernatant samples:
1) bed board: being added suitable DMEM culture medium containing 10%FBS for the MARC-145 cell that pancreatin has digested, and adjusts
Whole density is 1 × 105A/ml is added in 96 porocyte culture plates, cell is made to grow up to single layer；
2) it will collect in the DMEM culture medium of sterilizing EP Guan Zhongyong serum-free and connect containing virulent cell supernatant
Continuous 10 times of dilutions, from 10-1~10-10, each dilution will be sufficiently mixed uniformly；
3) it is successively inoculated into MARC-145 cell from high to low by dilution, each dilution is inoculated with 8 hole of a tandem, often
Hole is inoculated with 100 μ l, and taking two tandems is normal cell controls；
4) it is observed and recorded day by day since second day as a result, 5~7 days from being generally required；
5) TCID50 is calculated referring to Reed-Muench method.
As a result as shown in figure 4, when transfecting miR-c89 analogies postoperative infection GD-HD strain in PAMs cell, cell culture
Virus titer in supernatant has dropped 0.5-log and 1.2-log (P < 0.05) in 12hpi and pi for 24 hours respectively.
When the above results show that first transfect miR-c89 in PAMs infects HP-PRRSV again, miR-c89 can be significantly inhibited
The duplication of HP-PRRSV.
When first transfecting 50nM miR-c89 analogies in 2 PAMs cell of embodiment and then infecting low pathogenicity N-PRRSV,
The influence that miR-c89 replicates low pathogenicity N-PRRSV
(1) transfection of miR-c89 analogies and cell connect poison:
Bed board transfects, meets malicious (0.1MOI N-PRRSV CH1a) and receive sample with embodiment 1.
2.RT-qPCR detects PRRSV ORF7 mRNA relative expression levels
Extract RNA in cell, reverse transcription is cDNA and qPCR detection with embodiment 1.
As a result as shown in figure 5, transfecting the miR-c89 analogies postoperative infection N-PRRSV CH1a poison of 50nM in PAMs cell
When strain, the relative expression quantity of PRRSV ORF7 has dropped 96.6% compared with compareing analogies group in 12hpi and pi for 24 hours respectively
With 99.3%.
PRRSV genome copy numbers in 3.RT-qPCR detection assay Supernatant samples
RNA, reverse transcription, preparation positive criteria product (contain PRRSV ORF7 genetic fragment in extraction cell culture supernatant
Plasmid standard) and calculate in Supernatant samples PRRSV genome copy numbers with embodiment 1.
As a result as shown in fig. 6, transfecting miR-c89 analogies postoperative infection N-PRRSVCH1a strain 12hpi in PAMs cell
For 24 hours when pi, PRRSV genome copy numbers have dropped respectively compared with compareing analogies group in PAMs cell culture supernatant
59.7% and 97.6%.
4.TCID50 measures the titre of PRRS virus in Supernatant samples
Bed board, doubling dilution containing virulent cell supernatant, measurement, observation and are calculated with embodiment 1.
As a result as shown in fig. 7, transfecting miR-c89 analogies postoperative infection N-PRRSV CH1a strain in PAMs cell
When 12hpi and for 24 hours pi, the virus titer in cell culture supernatant had dropped respectively in 12hpi and pi for 24 hours 1.0-log and
2.4-log(P<0.05)。
When transfecting based on the above results it can be shown that first miR-c89 in PAMs and infect N-PRRSV again, miR-c89 can be shown
Write the duplication for inhibiting N-PRRSV.
When embodiment 3 transfects 50nM miR-c89 analogies after the highly pathogenic HP-PRRSV of PAMs cell infection, miR-
The influence that c89 replicates highly pathogenic HP-PRRSV
(1) cell connects poison and the transfection of miR-c89 analogies:
1) bed board is the same as embodiment 1；
2) it connects poison: HP-PRRSV GD-HD strain being inoculated with 0.01MOI after bed board 12h, according to formula PFU=number of cells
× MOI=0.7 × TCID50 calculates required virus liquid；24 orifice plates are taken out, after being cleaned with PBS, addition viral dilution, 37
DEG C culture；
3) transfect: transfection reagent is the X-tremeGENE siRNA Transfection Reagent of Roche Holding Ag, is pressed
It is transfected according to the operating procedure of transfection reagent；Transfection cocktail (analogies and siRNA are prepared after infecting 1h
Transfection reagent is dissolved in Opti-MEM respectively, then the two is mixed) incubation at room temperature 20min；By 24 orifice plates from
It is taken out in incubator, changes virus liquid into Opti-MEM, mixture is added dropwise and rocks mixing, is placed in cell incubator and trains
It supports；
4) it receives sample: 0h, 12h after infection, collecting cell sample and cell culture supernatant, -80 DEG C of preservations for 24 hours；Cell
Sample is used to detect the relative expression levels of PRRSV ORF7 gene in cell, and cell culture supernatant is discharged into training for detecting
Virus genomic copy number in nutrient solution supernatant.
(2) RT-qPCR detects PRRSV ORF7 mRNA relative expression levels:
Extract the RNA in cell, reverse transcription is that cDNA and qPCR is detected with embodiment 1.
As a result as shown in figure 8, transfecting miR-c89 analogies after infecting HP-PRRSV GD-HD strain in PAMs cell,
The relative expression quantity of PRRSV ORF7 has dropped 85.9% He compared with compareing analogies group in 12hpi and pi for 24 hours respectively
84.6%.
(3) PRRSV genome copy numbers in RT-qPCR detection assay Supernatant samples:
RNA, reverse transcription, preparation positive criteria product (contain PRRSV ORF7 genetic fragment in extraction cell culture supernatant
Plasmid standard) and calculate in Supernatant samples PRRSV genome copy numbers with embodiment 1.
As a result as shown in figure 9, transfecting miR-c89 analogies after infecting HP-PRRSV GD-HD strain in PAMs cell,
In 12hpi and pi for 24 hours, PRRSV genome copy numbers are distinguished compared with compareing analogies group in PAMs cell culture supernatant
Have dropped 97.8% and 99.7%.
(4) TCID50 measures the titre of PRRS virus in Supernatant samples
Bed board, doubling dilution containing virulent cell supernatant, measurement, observation and are calculated with embodiment 1.
The results are shown in Figure 10, transfects miR-c89 analogies, cell culture after GD-HD strain is infected in PAMs cell
Virus titer in supernatant has dropped 0.5-log and 0.3-log (P < 0.05) in 12hpi and pi for 24 hours respectively.
When the above results show to transfect 50nM miR-c89 analogies after infection HP-PRRSV in PAMs, miR-c89 can
Significantly inhibit HP-PRRSV duplication.
Embodiment 4 infects in PAMs when transfecting 50nM miR-c89 analogies after low pathogenicity N-PRRSV, miR-c89
Influence to low pathogenicity N-PRRSV duplication
(1) cell connects poison and the transfection of miR-c89 analogies:
Bed board connects malicious (0.01MOI N-PRRSV CH1a), transfection and receives sample with embodiment 3.
(2) RT-qPCR detects PRRSV ORF7 mRNA relative expression levels:
It extracts the RNA in cell, be reversed to cDNA and qPCR detection with embodiment 3.
As a result as shown in figure 11, miR-c89 analogies are transfected after N-PRRSV CH1a strain is infected in PAMs cell,
The relative expression quantity of PRRSV ORF7 has dropped 90.9% He compared with compareing analogies group in 12hpi and pi for 24 hours respectively
94.9%.
(3) PRRSV genome copy numbers in RT-qPCR detection assay Supernatant samples:
RNA, reverse transcription, preparation positive criteria product (contain PRRSV ORF7 genetic fragment in extraction cell culture supernatant
Plasmid standard) and calculate in Supernatant samples PRRSV genome copy numbers with embodiment 3.
As a result as shown in figure 12, miR-c89 analogies are transfected after N-PRRSV CH1a strain is infected in PAMs cell,
When 12hpi and for 24 hours pi, under PRRSV genome copy numbers are compared with compareing analogies group respectively in PAMs cell culture supernatant
65.3% and 81.9% have dropped.
(4) TCID50 measures the titre of PRRS virus in Supernatant samples
Bed board, doubling dilution containing virulent cell supernatant, measurement, observation and are calculated with embodiment 3.
As a result as shown in figure 13, miR-c89 analogies are transfected after N-PRRSV CH1a strain is infected in PAMs cell, carefully
Virus titer in born of the same parents' culture supernatant has dropped 0.71-log and 0.38-log (P < 0.05) in 12hpi and pi for 24 hours respectively.
When the above results show to transfect 50nM miR-c89 analogies after infection N-PRRSV in PAMs, miR-c89 can
Significantly inhibit N-PRRSV duplication.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Sequence table
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>a kind of pig source miR-c89 of anti-PRRSV infection and its application
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<170> SIPOSequenceListing 1.0
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<211> 21
<212> RNA
<213> Artificial Sequence
<400> 1
guacaguacu gugauaacug a 21
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<211> 55
<212> RNA
<213> Artificial Sequence
<400> 2
gguuaucaug guaccgaugc uguauaucug aaagguacag uacugugaua acuga 55
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 3
agatcatcgc ccaacaaaac 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
gacacaattg ccgctcacta 20
<210> 5
<211> 26
<212> DNA
<213> Artificial Sequence
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<210> 6
<211> 24
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Claims (7)
1. a kind of pig source miR-c89 sequence of anti-PRRSV infection, which is characterized in that the miR-c89 is nucleotide sequence coded
RNA sequence is as follows:
GUACAGUACUGUGAUAACUGA；SEQ ID NO.1.
2. a kind of pig source miR-c89 sequence of anti-PRRSV infection according to claim 1, which is characterized in that the pig source
Application of the miR-c89 in the drug or preparation of preparation prevention or treatment pig blue-ear disease.
3. a kind of pig source miR-c89 sequence of anti-PRRSV infection according to claim 2, which is characterized in that the miR-
C89 sequence transfection PAMs cell can inhibit duplication and proliferation of the PRRSV in PAMs cell.
4. a kind of precursor RNA of the pig source miR-c89 sequence of anti-PRRSV infection, which is characterized in that the miR-c89 nucleotide
The precursor RNA sequence of sequential coding is as follows:
GGUUAUCAUGGUACCGAUGCUGUAUAUCUGAAAGGUACAGUACUGUGAUAACUGA；SEQ ID NO.2.
5. the precursor of the miRNA of the pig source miR-c89 sequential coding of anti-PRRSV infection according to claim 4 a kind of
RNA, which is characterized in that the precursor RNA of the miRNA of the pig source miR-c89 sequential coding is in preparation prevention or treatment pig indigo plant ear
Application in the drug or preparation of disease.
6. the precursor RNA of the pig source miR-c89 sequential coding miRNA of anti-PRRSV infection according to claim 5 a kind of,
It is characterized in that, the precursor RNA transfection PAMs cell of the miRNA of the pig source miR-c89 sequential coding can inhibit PRRSV to exist
Duplication and proliferation in PAMs cell.
7. a kind of precursor RNA of the pig source miR-c89 sequence of anti-PRRSV infection according to claim 4, feature exist
In application of the precursor RNA of the miRNA of the pig source miR-c89 sequential coding in the gene modification pig for developing anti-blue otopathy.
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