CN101748124B - siRNA segment and application thereof used for curing and/or preventing Peste des petits ruminants - Google Patents
siRNA segment and application thereof used for curing and/or preventing Peste des petits ruminants Download PDFInfo
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Abstract
The invention provides a siRNA segment and application thereof used for curing and/or preventing Peste des petits ruminants. Both the siRNA segment and a carrier comprising the siRNA segment play the protection role on the Vero clones inflected by Peste des petits ruminants virus (PPRV); the siRNA segment and the carrier comprising the siRNA segment are discovered to be capable of reducing the mRNA level of PPRV-L protein by about 20 percent-40 percent through real-time quantitative PCR and immunoblot assay; therefore, the siRNA segment and the carrier comprising the siRNA segment of the invention can be used for preparing drugs used for curing and/or preventing Peste des petits ruminants, and have important values on curing Peste des petits ruminants with genes.
Description
Technical field
The present invention relates to a kind of siRNA fragment and application thereof, particularly relate to a kind of siRNA fragment and application thereof that is used for the treatment of and/or prevents PPR.
Background technology
RNA disturbs that (RNA interference RNAi) is sequence-specific gene silencing by double chain RNA mediate.In February, 1998, the Mello by graduate Fire of Washington Ka Naiji and University of Massachusetts Cancer center proposed this notion first in to the research of beautiful new rhabditis axei (Caenorhabditis elegan), and it all belongs to PTGS mechanism with coexpression of finding and elimination (quelling) phenomenon of finding in coarse arteries and veins spore mould in plant.This mechanism has been proved in nearly all eukaryotic cells such as fungi, Arabidopis thaliana, trypanosome, hydra, turbellarian worm, zebra fish and has all existed, even also has similar mechanism in intestinal bacteria.Also have this mechanism in finding mammalian cell after, RNAi is widely used in the treatment research of the research of gene function and tumour and virus disease as a kind of instrument of quick, effective, special inhibition of gene expression.
PPR virus (peste des petits ruminants virus, PPRV) be Paramyxoviridae Morbillivirus member, it is a kind of single minus-stranded rna virus of tunicary non-segmented negative, its virus particle is spherical in shape, 15948 Nucleotide of genome total length, comprise N-P/C/V-M-F-H-L totally 6 genes successively, the nucleoprotein (N albumen) of encoding respectively, L albumen cofactor (P albumen), underlying membrane albumen (M albumen), the fusion rotein (F albumen) that mediation peplos and host cell membrane merge, albumen (H albumen) as receptor protein and cell surface molecule bonded tool hemagglutinin activity, these six kinds of primary structure albumen of the albumen of the enzyme component of virus transcription enzyme and replicative enzyme (L albumen), and C, two kinds of Nonstructural Proteins of V.Described L albumen methylates, adds the activity of cap and polyadenylation in addition except having polymerase activity, be essential for duplicating of PPRV, and is very important in virus transcription and reproduction process.
PPR is a feature with heating, eye nasal discharge, stomatitis, diarrhoea and pneumonia, mainly causes small ruminant, especially goat and sheep morbidity, and morbidity and case fatality rate are all higher.PPR becomes one of important cause of disease that threatens livestock industry safety in many national eruption and prevalences at present.Though the vaccine of prevention PPRV has deactivation vaccine and weak malicious seedling; yet the use of vaccine is merely able to provide the part protection; make body clinical symptom no longer occur, can not stop subinfection again, and conventional vaccine needs could produce protection to body about fortnight usually.In addition, virulence also might take place and return strong phenomenon in weak malicious seedling strain under state of nature, causes the persistent infection of infecting flock of sheep, makes some conventional vaccines be difficult to obtain the ideal effect.Therefore, press for a kind of novel antiviral measure that more effective protection can be provided all PPRV virus strain of exploitation.
Summary of the invention
Therefore, the objective of the invention is to overcome existing treatment and the above-mentioned shortcoming and defect of preventing the PPR method, a kind of inhibition PPR virus genetic expression based on the RNAi technology is provided, thereby treats and/or prevents the specific RNA i fragment of PPR.
Above-mentioned purpose of the present invention realizes by the following technical solutions:
A kind of siRNA fragment that is used for the treatment of and/or prevents PPR, its sequence are any one following sequence:
L-462(SEQ?ID?NO.1)：
5’-GCACAUGCAUAGCUCUCAA-3’
3’-CGUGUACGUAUCGAGAGUU-5’；
L-5389(SEQ?ID?NO.2)：
5’-GCGUACAAGGAAGUUCUUA-3’
3’-CGCAUGUUCCUUCAAGAAU-5’。
Above-mentioned siRNA fragment can obtain by the method for chemosynthesis, in-vitro transcription, siRNA expression vector or siRNA expression framework.In preferred embodiments, hold the modification sequence that further adds 2-6 dT or 2-6 U, can reduce siRNA and in cell, degrade, strengthen its stability at described siRNA segmental 3 '.
In accordance with the following methods synthetic siRNA fragment is distinguished transfection to Vero clone (African green monkey kidney cell):
1, the cultivation of Vero cell: with the RPMI1640 substratum (GIBCO) that contains 10% foetal calf serum, in 37 ℃, 5%CO
2Cultivate under the condition;
2, transfection: adopt liposome Hiperfect (Qiagen) to carry out cell transfecting, with 1 μ gsiRNA transfection Vero cell (6 orifice plate).
Finish 24h after the transfection, inoculation PPRV (MOI=0.1) attacks poison.
After attacking malicious 24h, extract the cell total rna reverse transcription, as confidential reference items, detect the mRNA level of PPRV-L gene with the GAPDH gene, and pass through immunoblotting (Western Blot) and detect the proteic expression level of PPRV-L by real-time quantitative PCR (Real-time PCR).
The present invention also provides a kind of carrier that is used for the treatment of and/or prevents PPR, wherein comprises the siRNA fragment of one or both sequences for any one above-mentioned sequence.Described carrier is preferably retroviral vector (comprising lentiviral vectors), adenovirus carrier, gland relevant viral vector (AAV carrier) and plasmid vector etc., as pSuper carrier, pSilencer carrier, RNAi-Ready expression vector (ClonTech) etc.These carriers can be at eukaryotic cell, high-efficiency continuous expression siRNA in the mammalian cell especially, and itself or its product do not produce other stimulation or influence the body normal physiological function body, can be used for body safely.
SiRNA fragment and carrier thereof that the present invention is designed can be applicable to prepare the medicine that treats and/or prevents PPR.This medicine is for suppressing the medicine of PPR virus.Specifically, this medicine is for suppressing the medicine of PPR L protein expression.
The present invention is according to the strategy of siRNA design, searches (the GenBank number of landing: EU364809), design manyly to the siRNA fragment, and by the test of transfection Vero clone these siRNA fragments are carried out screening active ingredients of PPRV genome sequence from GenBank.The selection result shows, 2 pairs of siRNA fragments of the present invention's design can reduce the proteic expression of PPRV-L in the Vero cell, and wherein that the mRNA level of PPRV-L gene is reduced is about 40% for L-462, and L-5389 makes the mRNA level of PPRV-L gene reduce about 30%.The test of inserting carriers and transfection Vero clone with these two couples of siRNA shows, it is about 40% that the carrier that comprises L-462 reduces the mRNA level of PPRV-L gene, and the carrier that comprises L-5389 makes the mRNA level of PPRV-L gene reduce about 20%.Because PPRV-L albumen is essential for duplicating of PPRV, suppresses the proteic expression of PPRV-L and just suppressed growth and the breeding of PPRV in cell.SiRNA fragment L-462 that the present invention is designed or L-5389 descend the PPRV-L expressing quantity, can be applied to prepare the medicine that treats and/or prevents PPR.
In sum, the present invention is based on the RNAi technology, a kind of specific RNA i fragment of the PPRV-L of inhibition protein expression is provided and has comprised this segmental carrier, duplicate essential L protein gene expression by suppressing PPRV, thereby realize suppressing growth and the breeding of PPRV, can be applicable to prepare the medicine that treats and/or prevents PPR.
Description of drawings
Fig. 1 is that siRNA of the present invention is to the proteic mRNA level affects of PPRV-L result schematic diagram.
Fig. 2 is that siRNA of the present invention influences result schematic diagram to the proteic expression level of PPRV-L.
Fig. 3 is that recombinant plasmid of the present invention is to the proteic mRNA level affects of PPRV-L result schematic diagram.
Fig. 4 is that recombinant plasmid of the present invention influences result schematic diagram to the proteic expression level of PPRV-L.
Embodiment
Below the invention will be further described by specific embodiment.Should be understood that following examples only are used to illustrate the present invention, and be not used in the scope of the present invention that limits.
Employed technology in following examples comprises synthetic, the cell transfecting equimolecular biology techniques of gene sequencing, and cell cultures, detection technique etc., unless stated otherwise, is routine techniques known to those skilled in the art; Employed plant and instrument, reagent, clone etc., only this specification sheets specifies, is that those skilled in the art can be by public approach acquisition.
Embodiment 1: the design of RNAi sequence
Strategy according to the siRNA design: 1) begin to search ideal siRNA fragment from 50～100 Nucleotide in target gene initiator codon AUG downstream, the closer to 3 ' end of target gene, its gene silencing effect may be good more; 2) the siRNA fragment is preferably AA (Nn) UU (N represents any base, and n is a base number, between 19～29nt), and NA (Nn) UU and NA (Nn) NN sequence also can.3) has balanced base contents (being that G/C content is 30%～70%).According to PPRV Tibet strain isolated genome sequence (the GenBank number of landing: EU364809), design 3 pairs of siRNA fragments, its sequence is respectively:
L-462(SEQ?ID?NO.1)：
5’-GCACAUGCAUAGCUCUCAA-3’
3’-CGUGUACGUAUCGAGAGUU-5’；
L-5389(SEQ?ID?NO.2)：
5’-GCGUACAAGGAAGUUCUUA-3’
3’-CGCAUGUUCCUUCAAGAAU-5’；
L-1382(SEQ?ID?NO.3)：
5’-CCAUGUAUCUCAAAGACAA-3’
3’-GGUACAUAGAGUUUCUAUU-5’。
Above-mentioned siRNA fragment length is 21nt, and the mutant of minus strand and any one position thereof and known person genoid and genetic expression fragment do not have homology.
Further add 2-6 dT or 2-6 U modification sequence at the segmental 3 ' end of described siRNA, in cell, degrade, strengthen its stability to reduce siRNA.
In addition, the negative control of use and GAPDH confidential reference items sequence are respectively:
Negative control (SEQ ID NO.4):
5’-UUCUCCGAACGUGUCACGUTT-3’
3 '-TTAAGAGGCUUGCACAGUGCA-5 ' GAPDH confidential reference items (SEQ ID NO.5):
5’-GUAUGACAACAGCCUCAAGTT-3’
3’-TTCAUACUGUUGUCGGAGUUC-5’
Above-mentioned siRNA fragment all entrusts Shanghai lucky agate medicine biological technique company limited synthetic.
Embodiment 2: siRNA transfection Vero clone, and carry out PPRV and infect
Experiment material: Vero cell, the wild strain isolated of PPRV (MOI=0.1), PPRV polyclonal antibody.
1, transfection: the Vero cell is with RPMI 1640 substratum (GIBCO) that contain 10% foetal calf serum, in 37 ℃, 5%CO
2Cultivate under the condition.Adopt liposome Hiperfect (production of U.S. Qiagen company) to carry out cell transfecting, working method can be fully according to producer's explanation.During transfection, with 1 μ g siRNA transfection Vero cell (6 orifice plate).
2, PPRV infects: behind the transfection 24h, each culture plate adds PPRV cell toxicant (MOI=0.1), hatches 45min for 37 ℃, supplies substratum to volume of culture, continues at 37 ℃, 5%CO
2Cultivate 24h under the condition.
Embodiment 3: the detection of interference effect
I. real-time quantitative PCR analysis:
1. extract total RNA
Use Trizol reagent (Invitrogen) to extract cell total rna, step is with reference to Invitrogen company specification sheets.
The cell total rna that extracts is cDNA through mouse source ThermoScript II M-MLV (production of U.S. Promega company) with the reverse transcription of random hexamer primer, and step is with reference to Promega company specification sheets.
2. real-time quantitative PCR experiment
1) primer and probe design
With the positive control of baboon (Papio anubis) GAPDH gene as the real-time quantitative PCR reaction, design primer and probe sequence respectively, as shown in table 1.
Table 1 GAPDH gene and PPRV-L gene primer and probe sequence
2) reagent and instrument
Reagent: iron gram graceful (TaqMan) real-time quantitative PCR common reagent (Shanghai Ji Ma Bioisystech Co., Ltd);
Instrument: FTC-2000A real-time quantitative PCR instrument (maple mountain range, China).
3) reverse transcription goes out cDNA
Mix following reagent in the PCR pipe:
Oligo-dT 4μl
RNA 20μl
70 ℃ of insulation 10min, more than cooled on ice 2min, instantaneous centrifugal rapidly, add following reagent:
5×M-MLV?Buffer 8μl
dNTP(0.25mM) 4μl
RNasin 2μl
M-MLV 2μl
42 ℃ of insulations are after 2 hours, and in 70 ℃ of insulations 15 minutes, then cooled on ice obtained cDNA again.
4) PCR reaction system: as shown in table 2.
Table 2 PCR reaction system
| Composition | | Volume | |
| 2 * real-time quantitative PCR Master Mix | 1× | 10μl | |
| F primer (10 μ M) | 0.2μM | 0.4μl | |
| R primer (10 μ M) | 0.2μM | 0.4μl | |
| Probe (10 μ M) | 0.1μM | 0.2μl | |
| The cDNA template | — | 2μl | |
| Taq archaeal dna polymerase (5U/ μ l) | 0.5U/μl | 0.2μl | |
| dd?H 2O | Reach 20 μ l |
The real-time quantitative PCR reaction conditions: 95 ℃, the 5min sex change; 40 circulations: 95 ℃, 15 seconds; 55 ℃, 30 seconds; 72 ℃, 30 seconds.
5) experimental result:
Fig. 1 is that real-time quantitative PCR of the present invention detects the horizontal result schematic diagram of PPRV-L protein mRNA, and wherein contrast is the cell sample without the siRNA processing, and other processing is all consistent with sample.
C
TThe cycle number of being carried out when on behalf of the fluorescent signal in each reaction tubes, value reach preset threshold is got the C of various samples
TThe mean value of value calculates Δ C
T, Δ Δ C
TWith 2
-Δ Δ C T, concrete data processed result is as shown in table 3.
Table 3 data processed result
| The | ΔC | T | 2 -ΔΔC T | The t check |
| L-462 | 6.06±0.18 | 0.63(0.51～0.79) | P<0.05 | |
| L-1382 | 4.55±0.20 | 1.81(1.44～2.25) | P<0.05 | |
| L-5389 | 5.89±0.18 | 0.71(0.57～0.88) | P<0.05 | |
| Contrast | 5.40±0.25 | 1.00(0.79～1.28) | / |
Δ C
TRefer in the same sample the average C of gene to be checked and internal control gene GAPDH
TThe difference of value, i.e. Δ C
T=C
The T testing gene-C
The T internal control geneΔ Δ C
TThe Δ C that refers to all the other samples
TThe Δ C of value and control group corresponding gene
TWhat be worth is poor, i.e. Δ Δ C
T=(C
The T testing gene-C
The T internal control gene)
Experimental group-(C
The T testing gene-C
The T internal control gene)
Control group2
-Δ Δ C TBe according to Δ Δ C
TCalculating, is its fiducial interval in the bracket.
With 2
-Δ Δ C TThe expression of value representation experimental group testing gene is with respect to the variation multiple of control group, and the difference of L-462 and control group is: 1.00-0.63=0.37=37%; The difference of L-5389 and control group is: 1.00-0.71=0.29=29%.As seen, compare with control group, the relative expression quantity of L-462 and L-5389 is about 30-40%, and promptly the difference percentage of the relative wild-type group of L-462 and L-5389 expression amount reaches about 30-40%.
Carry out the T check through three repeated experiments, standard deviation is significant difference less than 0.05.
II. immunoblotting check
1. proteinic extraction
Use WIP histiocyte lysate capable (bio tech ltd of speeding is matched in Beijing) to extract protein, step is with reference to specification sheets.
2. proteinic polyacrylamide gel electrophoresis
1) preparation polyacrylamide gel, resolving gel concentration 12%, basic unit's gum concentration 5%, each component is as shown in table 4.
The preparation of table 4 polyacrylamide separation gel
| The reagent composition | Separation gel 12% (ml) | The reagent composition | Basic unit's glue 5% (ml) |
| Acry:Bis(30:0.8) | 6.00 | Acry:Bis(30:0.8) | 1.30 |
| 4×Tris·Cl/SDS，pH8.8 | 3.75 | 4×Tris·Cl/SDS，pH6.8 | 2.50 |
| H 2O | 5.25 | H 2O | 6.10 |
| 10% ammonium persulphate | 0.05 | 10% ammonium persulphate | 0.05 |
| TEMED | 0.01 | TEMED | 0.01 |
2) in Eppendorf tube, press 1:1 (v/v) dilution testing protein quality sample with 2 * SDS sample loading buffer, boil 5min in 100 ℃.Sample 40 μ l on the every hole of sample, protein molecular weight standard thing (Marker) is gone up sample 20 μ l.
3) use 1 * SDS electrophoretic buffer, 120V voltage electrophoresis 2 hours.
4) gel slab is taken out, shift out gel, the mark gel is so that identification application of sample order.
3. protein is transferred to nitrocellulose filter from sds page
1), cuts 6 filter paper and 1 nitrocellulose filter according to the gel size.Nitrocellulose filter is floated on soak on the water surface of a dish deionized water more than 5 minutes and stay bubble on filter membrane with expeling.In a shallow pallet, add a small amount of transfering buffering liquid, 6 filter paper are soaked in wherein.
Transfering buffering liquid: 190mmol/L glycine, 25mmol/L Tris alkali, 20% methyl alcohol.
2) transfer device is installed in the following order:
Filter paper → electrode (anode) that soaked with transfering buffering liquid filter paper → gel → nitrocellulose filter → 3 that soaked with transfering buffering liquid bottom electrode (negative electrode) → sponge pad → 3.
3) press the 300mA making current according to the gel area, electrotransfer finished in 2 hours.Keep low temperature in the electrotransfer process.
4. the protein that is fixed on the nitrocellulose filter is dyeed
1) treats the nitrocellulose filter drying, it is floated on the water surface of a dish deionized water, make filter membrane moistening from bottom to top by wicking action.Then filter membrane is soaked in the water, soaks more than 5 minutes with expeling and stay thereon bubble.
2) filter membrane is transferred to contained in the pallet that Ponceau S uses liquid dyeing 5-10 minute, shake dye liquor therebetween gently.
Mix following ingredients, be made into Ponceau S stock solution (10 *):
Ponceau S 2g
Trichoroacetic acid(TCA) 30g
Sulphosalicylic acid 30g
Add water to 100ml
Add 9 parts of deionized waters with 1 part of above-mentioned stock solution and promptly be made into Ponceau S use liquid, promptly discarded after the use.
3) after protein band occurs, under room temperature, use the rinsed with deionized water nitrocellulose filter, change water number therebetween.
4) mark as the proteic position of the reference of molecular weight standard with pencil.
5. seal the immunoglobulin (Ig) binding site of nitrocellulose
1) nitrocellulose filter is put into the plastics bag that can add heat sealing, according to the filter membrane area with 0.1ml/cm
2Amount add confining liquid (8% skimmed milk), get rid of bubble, airtight sack lay on the shaker platform of shaking gently under room temperature incubation 1 hour.
2) cut off plastics bag, discard confining liquid, add together incubation of anti-target protein antibody-solutions and filter membrane immediately.
6. the combination of antibody and target protein
Adding one resists:
1) in the plastics bag that the nitrocellulose filter of handling with aforesaid method is housed, presses 0.1ml/cm
2Amount add confining liquid and an amount of first antibody (PPRV polyclonal antibody, 20 μ l/5ml).
2) seal sack after getting rid of bubble, filter membrane is lain on the shaker platform of shaking gently, in 37 ℃ of following incubations 1～2 hour.
3) cut off plastics bag, discarded confining liquid and antibody are used 25ml PBS rinsing filter membrane 3 times, each 10 minutes.
4), use together incubation of secondary immunoreagent and filter membrane immediately by the method for introducing below.
Adding two resists:
1) after the last washing of PBS, nitrocellulose filter is transferred in the pallet that PBS solution is housed, under room temperature, shaken incubation gently 10 minutes.
2) filter membrane is transferred in the plastics bag that can add heat sealing, presses the filter membrane area and add 0.1ml/cm
2Confining liquid.
3) add enzyme di-level reagent (HRP-goat anti-rabbit igg, 20 μ l/5ml), seal.
4) under 37 ℃, shake gently, filter membrane and enzyme di-level reagent incubation 40min together.
5) filter membrane is transferred to TBST solution.Under room temperature, shake incubation 10 minutes gently.Repeat 3 times, at every turn the TBST solution that more renews.
7.ECL chemical luminous substrate detects
0.5ml buffer A+0.5ml buffer B joins on the NC film, hatches 5min, the 0.5min of X-ray sheet reaction then, and developing solution is handled 0.5min, and stop bath is handled 0.5min, takes pictures.Fig. 2 detects PPRV-L protein expression level result schematic diagram for immunoblotting, and wherein the cell sample of contrast for not using siRNA to handle is all consistent with sample L-462, L-1382, L-5389 to other processing of this sample.
As seen from the figure, the band of L-462 and L-5389 is obviously light in contrast, and the band of L-1382 is obviously deep, this figure can be qualitatively as the reference of real-time quantitative RT-PCR.
Embodiment 4: siRNA is imported carrier
I. oligonucleotide design
Select tangible L-462 of interference effect and L-5389 sequence for use, the siRNA sequences Design is similar in the design and implementation example 1, on the basis of former siRNA sequence (being converted to dna sequence dna), design, add the BglII restriction enzyme site at its first chain, 5 ' end, second chain, 5 ' end adds the HindIII restriction enzyme site, obtain two sections complementary oligonucleotide fragments, its sequence is:
L-462：
5’-
GATCCCCGCACATGCATAGCTCTCAA
TTCAAGAGATTGAGAGCTATGCATGTGC
TTTTTA-3’
3’-
GGGCGTGTACGTATCGAGAGTT
AAGTTCTCTAACTCTCGATACGTACACG
AAAAATTCGA-5’
L-5389：
5’-
GATCCCCGCGTACAAGGAAGTTCTTA
TTCAAGAGATAAGAACTTCCTTGTACGC
TTTTTA-3’
3’-
GGGCGCATGTTCCTTCAAGAAT
AAGTTCTCTATTCTTGAAGGAACATGCG
AAAAATTCGA-5’
Above-mentioned oligonucleotide fragment all entrusts Invitrogen company synthetic.
II. the structure of carrier
H1-RNA promotor on the pBabe-Puro carrier is cloned in the promoterless shuttle vectors pSuper carrier (Oligoengine), the new shuttle vectors called after pBabe-Super that obtains, this carrier can start the expression of siRNA in recombinant adenovirus.
Above-mentioned synthetic annealing oligonucleotide also is connected to BglII and the HindIII site of pBabe-Super, the plasmid that obtains building, called after pBabe-L-462 and pBabe-L-5389, through the EcoRI enzyme cut evaluation, order-checking is identified correct.
Embodiment 5: the detection of recombinant plasmid interference effect
I. use recombinant plasmid transfection Vero cell, and infect PPRV, concrete grammar is referring to embodiment 2.
II. real-time quantitative PCR analysis, concrete grammar is referring to embodiment 3.
The result as shown in Figure 3.Fig. 3 analyzes recombinant plasmid of the present invention to the proteic mRNA level affects of PPRV-L result schematic diagram for real-time quantitative PCR.Wherein contrast is not for importing the cell sample that siRNA handles, and other processing is all consistent with sample.Concrete Data Processing in Experiment result is as shown in table 5.
Table 5 data processed result
| The | ΔC | T | 2 -ΔΔC T | The t check |
| pBabe-L-462 | 6.05±0.17 | 0.59(0.50～0.69) | P<0.05 | |
| pBabe-L-5389 | 5.59±0.13 | 0.81(0.70～0.93) | P<0.05 | |
| Contrast | 5.28±0.15 | 1.00(0.86～1.16) | / |
With 2
-Δ Δ C TThe expression of value representation experimental group testing gene is with respect to the variation multiple of control group, and the difference of pBabe-L-462 and control group is: 1.00-0.59=0.41=41%; The difference of pBabe-L-5389 and control group is: 1.00-0.81=0.19=19%.As seen, compared with the control, the relative expression quantity of pBabe-L-462 and pBabe-L-5389 is about 19-41%, and promptly plasmid pBabe-L462 and pBabe-L5389 reach about 19-41% with respect to the difference percentage of control group expression amount.
Carry out the T check through three repeated experiments, standard deviation is significant difference less than 0.05.
III. immunoblotting check, concrete grammar is referring to embodiment 3
The result as shown in Figure 4.Recombinant plasmid of the present invention is to PPRV-L protein expression level result schematic diagram for immunoblotting detects for Fig. 4, and wherein contrast is not for importing the cell sample that siRNA handles, and other processing is all consistent with sample.As seen from the figure, the L expressing quantity of recombinant plasmid pBabe-L-462 and pBabe-L-5389 is less than the proteic expression amount of control group M, shows that these two kinds of recombinant plasmids have the inhibition effect to the M genetic transcription protein expression of PRRSV.This figure can be qualitatively as the reference of real-time quantitative RT-PCR.
Sequence table
＜110〉Institute of Zoology, Academia Sinica
＜120〉be used for the treatment of and/or prevent the siRNA fragment and the application thereof of PPR
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Claims (11)
1. siRNA fragment that is used for the treatment of and/or prevents PPR, its sequence is any one following sequence:
L-462：
5’-GCACAUGCAUAGCUCUCAA-3’
3’-CGUGUACGUAUCGAGAGUU-5’
L-5389：
5’-GCGUACAAGGAAGUUCUUA-3’
3’-CGCAUGUUCCUUCAAGAAU-5’。
2. siRNA fragment according to claim 1 is characterized in that, the modification sequence of the segmental 3 ' end of described siRNA is 2-6 dT or 2-6 U.
3. carrier that is used for the treatment of and/or prevents PPR wherein comprises the siRNA fragment of sequence for any one following sequence:
L-462：
5’-GCACAUGCAUAGCUCUCAA-3’
3’-CGUGUACGUAUCGAGAGUU-5’
L-5389：
5’-GCGUACAAGGAAGUUCUUA-3’
3’-CGCAUGUUCCUUCAAGAAU-5’。
4. carrier according to claim 3 is characterized in that, the modification sequence of the segmental 3 ' end of described siRNA is 2-6 dT or 2-6 U.
5. according to claim 3 or 4 described carriers, it is characterized in that described carrier is retroviral vector, adenovirus carrier, gland relevant viral vector and plasmid vector etc.
6. carrier according to claim 5 is characterized in that, described carrier is pSuper carrier, pSilencer carrier and RNAi-Ready expression vector.
7. carrier according to claim 5 is characterized in that described retroviral vector comprises lentiviral vectors.
8. siRNA fragment according to claim 1 treats and/or prevents application in the medicine of PPR in preparation.
9. carrier according to claim 3 treats and/or prevents application in the medicine of PPR in preparation.
10. according to Claim 8 or 9 described application, it is characterized in that the described medicine of PPR that treats and/or prevents is for suppressing the medicine of PPR virus.
11. according to Claim 8 or 9 described application, it is characterized in that the described medicine of PPR that treats and/or prevents is for suppressing the medicine of PPR virus L protein expression.
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| CN102851293B (en) * | 2011-10-26 | 2013-07-17 | 中国农业科学院兰州兽医研究所 | Sequence siRNA-165 capable of targeted inhibition of capripoxvirus ORF095 gene |
| CN102827842B (en) * | 2011-10-26 | 2013-06-05 | 中国农业科学院兰州兽医研究所 | Sequence siRNA-296 (small interfering Ribonucleic Acid-296) for restraining sheep pox virus ORF095 gene in targeted way |
| CN102417907B (en) * | 2011-10-26 | 2013-01-02 | 中国农业科学院兰州兽医研究所 | SiRNA (small interfering RNA) sequences of targeted inhibition capripoxvirus ORF095 genes |
| CN106591373B (en) * | 2016-12-23 | 2020-03-24 | 中国农业科学院兰州兽医研究所 | Preparation method of sensitive cell subcloned Vero/Slam for enhancing PPRV replication |
| CN106755110B (en) * | 2016-12-23 | 2020-09-01 | 中国农业科学院兰州兽医研究所 | Preparation method of sensitive cell subcloned Vero/Slam/V for enhancing PPRV replication |
| CN113416768B (en) * | 2021-07-06 | 2022-05-17 | 中国农业科学院兰州兽医研究所 | Application of PRKRA gene as target in inhibiting replication of peste des petits ruminants virus |
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