CN1884526A - RNA interference method for specificly and high effectively treating CSFV infection and biological formulation - Google Patents

Abstract

The invention provides a specific and high-performance interferometric technique for treating RNA infected by CSFV, which designs specific siRNA sequence for dissimilar genes of hog cholera virus and obtains high-performance and specific interfering RNA molecule for reducing hog cholera virus by T7 RNA polyase external rerecording system and plasmid expression system, so is suitable for commercial manufacture. The biological agent produced by this method has 92.9-99.0% high depression efficiency and can appreciably prevent CSFV affection inside sensitized animals to make the animals relieve from morbidity and death.

Description

A kind of special, efficiently treat RNA interference method and the biotechnological formulation that CSFV infects

Technical field

The invention provides-kind special, efficiently treat the RNA interference method that CSFV infects, the biotechnological formulation that utilizes this method to produce also is provided simultaneously, relate to the medicine for the treatment of swine fever virus infection, belong to biological pharmacy technical field.

Background technology

Write a Chinese character in simplified form CSFV below the Pestivirus suis that the present invention relates to.

At present, in order to prevent the general employing passive immunization (injection hyper-immune serum) and the active immunity method of swine fever virus infection, it is popular still still can not to prevent and treat swine fever fully.

RNA disturbs (RNA interference, RNAi) reported first was in 1998, it is a kind of brand-new technology that just grew up gradually in nearly 2 years, RNAi is considered to the virological investigation field that is used in the lump of the most effectual way of anti-virus infection since the great attention a person that finds to be subjected to the virological investigation.The key of using RNAi technology inhibition virus multiplication is to obtain efficient, the special little RNA of interference (siRNA) sequence.The medicine of using RNA interference method production for treating swine fever virus infection does not by retrieval appear in the newspapers.

Summary of the invention

The invention provides a kind of special, efficiently treat the RNA interference method that CSFV infects, by designing at the heterogeneic special siRNA sequence of Pestivirus suis, utilize T7 RNA polymerase in-vitro transcription system and plasmid expression system, obtain the disturbance RNA molecule of efficient, special inhibition Pestivirus suis, be applicable to suitability for industrialized production.

The present invention also provides the biotechnological formulation that utilizes this side to produce, and up to 92.9-99.0%, can obviously stop the infection of CSFV to the inhibition efficient of CSFV in responsive animal body, makes animal avoid morbidity and dead.

Technical solution of the present invention may further comprise the steps:

1. select the sequence of disturbance RNA molecule effect:

After utilizing the synthetic rnai molecule of RNA perturbation technique design, swine fever virus infection there is obvious therapeutic action thereby these rnai molecules can special, efficiently suppress Pestivirus suis propagation.

Conservative region in the Pestivirus suis genome sequence is according to having AA-N ₁₉The 21nt sequence of feature is carried out homology and secondary structure analysis, selects following 11 the disturbance RNA molecule effect sequences on the Pestivirus suis genome:

Table 1 disturbance RNA molecule effect sequence and position thereof

At gene	SiRNA molecule interference sequence	The position *	Name
				Npro	AAGGATAGGTAGGGTGACAGG	721-741	N1
Npro	AAGAACCCTGAAGTGGATTAG	820-840	N2
				C	AAGTGGAAGTAAAGAGAAGAA	892-912	S6
E rns	AAGAATAAACCACCAGAATCT	1082-1102	S7
				E1	AAGGTGGCTTTGTTATTTGAA	2255-2275	S8
NS2-3	AAGATGACTAATGGACTTAGT	3923-3943	S10
				NS4A	AAGAGGCATATACCAGTAGTCA	7253-7274	S11
NS4B	AAGCTGGAATTACAATAATCT	7837-7857	S2
				NS5A	AAGATGAAGGCAGTTAAGAAT	8606-8626	S3
NS5B	AAGAATGAGAAGAGGGACGTC	10700-10720	5B1
				3’UTR	AAGTTACCACACTACACTCAT	12205-12225	S12

*: the position relatively with reference to strain shimen, GenBank accession number: AF092448.

2. make swine fever treatment biotechnological formulation according to disturbance RNA molecule effect sequence:

Can make by following two kinds of methods

1) external synthetic siRNA molecular biosciences preparation:

According to positive-sense strand and the antisense strand template DNA and the T7 RNA polymerase binding sequence of disturbance RNA molecule effect sequence difference composite coding siRNA molecule, and definite control sequence is the reverse sequence of N1 sequence.

Sequence is as follows:

T7 RNA polymerase binding sequence: GGATCCTAATACGACTCACTATA

5B1 positive-sense strand template sequence: AAGACGTCCCTCTTCTCATTCTATAGTGAGTCGTATTAGGATCC

5B1 antisense strand template sequence: AAGAATGAGAAGAGGGACGTCTATAGTGAGTCGTATTAGGATCC

N1 positive-sense strand template sequence: AACCTGTCACCCTACCTATCCTATAGTGAGTCGTATTAGGATCC

N1 antisense strand template sequence: AAGGATAGGTAGGGTGACAGGTATAGTGAGTCGTATTAGGATCC

N2 positive-sense strand template sequence: AACTAATCCACTTCAGGGTTCTATAGTGAGTCGTATTAGGATCC

N2 antisense strand template sequence: AAGAACCCTGAAGTGGATTAGTATAGTGAGTCGTATTAGGATCC

S6 positive-sense strand template sequence: AATTCTTCTCTTTACTTCCACTATAGTGAGTCGTATTAGGATCC

S6 antisense strand template sequence: AAGTGGAAGTAAAGAGAAGAATATAGTGAGTCGTATTAGGATCC

S7 positive-sense strand template sequence: AAAGATTCTGGTGGTTTATTCTATAGTGAGTCGTATTAGGATCC

S7 antisense strand template sequence: AAGAATAAACCACCAGAATCTTATAGTGAGTCGTATTAGGATCC

S8 positive-sense strand template sequence: AATTCAAATAACAAAGCCACCTATAGTGAGTCGTATTAGGATCC

S8 antisense strand template sequence: AAGGTGGCTTTGTTATTTGAATATAGTGAGTCGTATTAGGATCC

S10 positive-sense strand template sequence: AAACTAAGTCCATTAGTCATCTATAGTGAGTCGTATTAGGATCC

S10 antisense strand template sequence: AAGATGACTAATGGACTTAGTTATAGTGAGTCGTATTAGGATCC

S11 positive-sense strand template sequence: AATGACTACTGGTATATGCCTCTATAGTGAGTCGTATTAGGATCC

S11 antisense strand template sequence: AAGAGGCATATACCAGTAGTCATATAGTGAGTCGTATTAGGATCC

S12 positive-sense strand template sequence: AAATGAGTGTAGTGTGGTAACTATAGTGAGTCGTATTAGGATCC

S12 antisense strand template sequence: AAGTTACCACACTACACTCATTATAGTGAGTCGTATTAGGATCC

S2 positive-sense strand template sequence: AAAGATTATTGTAATTCCAGCTATAGTGAGTCGTATTAGGATCC

S2 antisense strand template sequence: AAGCTGGAATTACAATAATCTTATAGTGAGTCGTATTAGGATCC

S3 positive-sense strand template sequence: AAATTCTTAACTGCCTTCATCTATAGTGAGTCGTATTAGGATCC

S3 antisense strand template sequence: AAGATGAAGGCAGTTAAGAATTATAGTGAGTCGTATTAGGATCC

The positive-sense strand template sequence of contrast: AACCTATCCATCCCACTGTCCTATAGTGAGTCGTATTAGGATCC

The antisense strand template sequence of contrast: AAGGACAGTGGGATGGATAGGTATAGTGAGTCGTATTAGGATCC

It is 100pmol/L that above-mentioned synthetic dna sequence dna is diluted respectively with the water of no DNA enzyme, no RNA enzyme, with PCR method they is synthesized double chain DNA sequence respectively:

The T7 RNA polymerase binding sequence DNA 5.0 μ L that in the PCR pipe of the no DNA enzyme of cleaning, no RNA enzyme, add dilution, the positive-sense strand and the antisence strand dna 5.0 μ L that add above-mentioned siRNA respectively, 10 * dna polymerase buffer liquid, 5.0 μ L, 10mmol/L dNTP 1.0 μ L, water 33.8 μ L, place 95 ℃ of sex change 2min of PCR instrument, add pfu archaeal dna polymerase 0.2 μ L (1U), again in 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 10s, after 5 circulations, extend 7min after 72 ℃.After reaction finishes, adding is handled and autoclaved 3mol/L sodium-acetate (pH 5.2) 5.0 μ L through DEPC, the dehydrated alcohol that adds 2.5 times of volumes then, place 10min, the centrifugal 10min of 12000r/min, abandoning supernatant in-20 ℃ behind the mixing, precipitate with 70% washing with alcohol, be deposited in drying at room temperature 10min, add the abundant dissolution precipitation of water 50 μ L of no DNA enzyme, no RNA enzyme, the polyacrylamide gel electrophoresis checks that template DNA prepares the result.

Dubbing method is pressed T7 RiboMAX ^TMExpress RNAi System specification sheets carries out in-vitro transcription: add the template DNA 4.0 μ L of above-mentioned preparation successively in the PCR pipe of the no DNA enzyme of cleaning, no RNA enzyme, RiboMAX ^TMExpress T7 2 * damping fluid 10.0 μ L, the water 4.0 μ L of nuclease free, Enzyme Mix, T7 Express 2.0 μ L place 37 ℃ of reaction 1h then; The DNA enzyme 1.0 μ L (1U) that add no RNA enzyme, 37 ℃ of 30min; Mix corresponding siRNA justice and antisense strand transcript, 70 ℃ of insulation 10min slowly are cooled to room temperature; The 3mol/L sodium-acetate (pH 5.2) that adds 1/10 volume, add isopyknic Virahol, ice bath 5min behind the mixing, the centrifugal 10min of 13000r/min, the careful suction removed supernatant liquor, 500 μ L, 70% washing with alcohol precipitation, be deposited in drying at room temperature 10-15min, add the abundant dissolution precipitation of 100 μ L water, electrophoretic examinations is packed as concentration 0.3 μ g/ μ L ,-80 ℃ of preservations behind the mensuration content, the siRNA molecular water solution that obtains the in-vitro transcription generation is swine fever treatment biotechnological formulation, according to the sequence name nominating is respectively: siN1, siN2, siS6, siS7, siS8, siS10, siS11, siS12, siS2, siS3, si5B1, the si contrast.

2) biotechnological formulation of plasmid expression shRNA disturbing molecule:

According to the requirement of pSilencer3.1H1 Hygro (Ambion) shRNA carrier design, be designed for and express shRNA interferential dna sequence dna, layout strategy is seen Fig. 2.In order to improve the efficient that the correct annealing of sequence becomes double chain DNA molecule, be designed to strategy with archaeal dna polymerase synthetic PCR, the dna sequence dna of expressing purpose shRNA sequence is split as the sequence of two sections part complementary pairings, use the VNTI3.0 assistant analysis then, making the complementary pairing base between two sections sequences of fractionation is 21-23bp, be less than 10bp and respectively split sequence self pairing base, to improve the efficient of the synthetic double-stranded template DNA of PCR.

Table 2 synthesizes each shRNA template DNA sequence

Name	The template DNA sequence
		N1	GACGGATCCGGATAGGTAGGGTGACAGGTTCAAGAGACCTGTC
TGCAAGCTTTTCCAAAAAAGGATAGGTAGGGTGACAGGTCTCTTGAACCTGT
	N2		GACGGATCCGAACCCTGAAGTGGATTAGTTCAAGAGACTAATC
TGCAAGCTTTTCCAAAAAAGAACCCTGAAGTGGATTAGTCTCTTGAACTAATC
		S6	GGAGGATCCCGTGGAAGTAAAGAGAAGAATTCAAGAGATTCTTC
CCGAAGCTTTTCCAAAAAAGTGGAAGTAAAGAGAAGAATCTCTTGAATTCTTCTC
	S7		GGAGGATCCCGAATAAACCACCAGAATCTTTCAAGAGAAGATTC
CCGAAGCTTTTCCAAAAAAGAATAAACCACCAGAATCTTCTCTTGAAAGATTCTG
		S8	GGAGGATCCCGGTGGCTTTGTTATTTGAATTCAAGAGATTCAA
CCGAAGCTTTTCCAAAAAAGGTGGCTTTGTTATTTGAATCTCTTGAATTCAAAT
	S10		GGAGGATCCCGATGACTAATGGACTTAGTTTCAAGAGAACTAAG
CCGAAGCTTTTCCAAAAAAGATGACTAATGGACTTAGTTCTCTTGAAACTAAGTC
		S11	GGAGGATCCCGAGGCATATACCAGTAGTCATTCAAGAGATGACTAC
CCGAAGCTTTTCCAAAAAAGAGGCATATACCAGTAGTCATCTCTTGAATGACTAC

S2	GGAGGATCCCGCTGGAATTACAATAATCTTTCAAGAGAAGATTATTG
		CCGAAGCTTTTCCAAAAAAGCTGGAATTACAATAATCTTCTCTTGAAAGATT
	S3		GGAGGATCCCGATGAAGGCAGTTAAGAATTTCAAGAGAATTC
CCGAAGCTTTTCCAAAAAAGATGAAGGCAGTTAAGAATTCTCTTGAAATTCTTAAC
		5B1	GACGGATCCGAATGAGAAGAGGGACGTCTTCAAGAGAGACGTCC
TCGAAGCTTTTCCAAAAAAGAATGAGAAGAGGGACGTCTCTCTTGAAGACG
	S12		GGAGGATCCCGTTACCACACTACACTCATTTCAAGAGAATGAGTG
CCGAAGCTTTTCCAAAAAAGTTACCACACTACACTCATTCTCTTGAAATGAGTG
		Contrast	GACGGATCCGGACAGTGGGATGGATAGGTTCAAGAGACCTATC
GGCAAGCTTTTCCAAAAAAGGACAGTGGGATGGATAGGTCTCTTGAACCTATCC

Concrete steps are as follows:

With the dilution of synthetic single stranded DNA is 100 μ mol/L, with PCR method they are synthesized double-stranded DNA respectively: get the DNA 5.0 μ L of dilution, add 10 * dna polymerase buffer liquid, 5.0 μ L, 10mmol/L dNTP1.0 μ L, water 33.8 μ L, place 95 ℃ of sex change 2min of PCR instrument, add pfu archaeal dna polymerase 0.2 μ L (1U), again in 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 10s after 5 circulations, extend 7min after 72 ℃.After reaction finishes, add 3mol/L sodium-acetate (pH 5.2) 5.0 μ L, the dehydrated alcohol that adds 2.5 times of volumes is then placed 10min, the centrifugal 10min of 12000r/min in-20 ℃ behind the mixing, abandoning supernatant, with 1000 μ L, 70% washing with alcohol precipitation, abandoning supernatant is deposited in drying at room temperature 10min, add the abundant dissolution precipitation of 50 μ L water ,-80 ℃ of preservations are standby.

The DNA that expresses shRNA with BamHI and HindIII double digestion inserts fragment, enzyme is cut digestion and is finished the back in 70 ℃ of insulation 15min deactivation restriction endonucleases, phenol/chloroform extracting, respectively they are cloned into the BamHI and the HindIII site of pSilencer3.1H1 Hygro carrier behind the ethanol sedimentation, transformed into escherichia coli, carry out PCR Rapid identification with 31H1FP (GTGCTGCAAGGCGATTAAGTTGG) and 31H1RP (GCGGATAACAATTTCACACAGG) for primer to transforming bacterium colony, the PCR positive colony is extracted plasmid in a small amount, the aqueous solution that obtains plasmid is swine fever treatment biotechnological formulation, respectively according to the sequence title with these plasmid called after: shN1, shN2, shS6, shS7, shS8, shS10, shS11, shS12, shS2, shS3, sh5B1, the sh contrast.

Have the effect that suppresses Pestivirus suis propagation by following detection method proof preparation of the present invention:

Example 1

External synthetic siRNA suppresses Pestivirus suis propagation:

1. count the PK-15 cell, with containing the MEM nutritive medium of 10% calf serum, 0.3% glutamine, antibiotic-free with 1.0 * 10 ⁴Go down to posterity in 24 porocyte culture plates in/hole, 37 ℃ of 5%CO ₂Cultivate 24h, make attached cell growth full scale be about 30%-50%, inhale and remove nutritive medium, use the MEM nutritive medium of serum-free antibiotic-free to wash monolayer cell 3 times, the MEM that adds 450 μ L serum-free antibiotic-frees is standby.

2. use X-tremeGene siRNA Transfection Reagent (Roche) with synthetic siRNA molecule transfection PK-15 cell, concrete grammar is: respectively at the MEM nutritive medium 47.5 μ L that add the serum-free antibiotic-free in the centrifuge tube of nuclease free, X-tremeGene siRNA Transfection Reagent 2.0 μ L, mixing gently, room temperature leaves standstill 3-5min, get the centrifuge tube of another nuclease free, the MEM nutritive medium 48.0 μ L that add the serum-free antibiotic-free, siRNA molecule 2.0 μ L, mix gently, room temperature leaves standstill 3-5min, mix solution in above-mentioned two centrifuge tubes, room temperature leaves standstill 15-20min, make liposome and siRNA form transfection composite, add respectively then in the PK-15 cell monolayer, gently mixing, hatch 4-6h for 37 ℃, with the MEM of serum-free antibiotic-free washing transfectional cell, use instead contain 2% calf serum the MEM nutritive medium in 37 ℃, 5%CO ₂Incubator is cultivated 20h, with 5.0 * 10 ²TCID ₅₀The Pestivirus suis Shimen strain cell of siRNA that infected transfection, infect the back and cultivate 72h, with the half cell infection amount (TCID of indirect immunofluorescence assay, Real-time PCR and virus ₅₀) method such as mensuration detects the propagation of Pestivirus suis in transfectional cell, measures the restraining effect of siRNA to viral gene expression in the swine fever virus infection breeding.

Table 1 indirect immunofluorescence assay detects the infected situation result of cell

The siRNA molecule	The indirect immunofluorescence result
		The si contrast	Cell 100% is infected
siN1	Cells infected is less than 1%
		siN2	Cells infected is less than 1%
siS2	Cells infected is less than 5%
		siS3	Cells infected is less than 5%
siS6	Cells infected is less than 5%
		siS7	Cells infected is less than 5%
siS8	Cells infected is less than 5%
		siS10	Cells infected is less than 5%
siS11	Cells infected is less than 5%
		siS12	Cells infected is less than 5%
si5B1	Cells infected is less than 1%

After the PK-15 cell infection Pestivirus suis of transfection siRNA, the indirect immunofluorescence detected result shows, the PK-15 cell of the PK-15 cell of untransfected and transfection si contrast, behind the pestivirus 72h of infected pigs, nearly all cell all can be by fluorescent antibody staining, and cell sends green fluorescence, after the cell infection Pestivirus suis of the siRNA that the transfection Pestivirus suis is special, have only few cell can be by fluorescent antibody staining, the cell that fluorescence occurs be less than 1% of whole cells.

Table 2Real-time PCR method detects viral genome propagation situation

The siRNA molecule	Viral genome propagation behind the infective virus 72h	Ratio with contrast
			The si contrast	5.95×10 5The total RNA of copies/5ng	-
siN1	4.89×10 4The total RNA of copies/5ng	1/12.1
			siN2	1.35×10 5The total RNA of copies/5ng	1/4.4
siS2	1.82×10 5The total RNA of copies/5ng	1/3.3
			siS3	2.13×10 5The total RNA of copies/5ng	1/2.8
siS6	1.53×10 5The total RNA of copies/5ng	1/3.9
			siS7	2.86×10 5The total RNA of copies/5ng	1/2.1
siS8	1.94×10 5The total RNA of copies/5ng	1/3.1
			siS10	1.60×10 5The total RNA of copies/5ng	1/3.7
siS11	2.03×10 5The total RNA of copies/5ng	1/2.9
			siS12	1.89×10 5The total RNA of copies/5ng	1/3.2
si5B1	1.40×10 5The total RNA of copies/5ng	1/4.2

The result shows: 72h after the cell infection virus of transfection contrast siRNA molecule, the Pestivirus suis geneome RNA copy number in the cell total rna is 5.95 * 10 ⁵The total RNA of copies/5ng, and the Pestivirus suis geneome RNA copy number of siN1, siN2, si5B1, siS2, siS3, siS6, siS7, siS8, siS10, siS11, siS12 transfectional cell is respectively 4.89 * 10 ⁴, 1.35 * 10 ⁵With 1.40 * 10 ⁵, 1.82 * 10 ⁵, 2.13 * 10 ⁵, 1.53 * 10 ⁵, 2.86 * 10 ⁵, 1.94 * 10 ⁵, 1.60 * 10 ⁵, 2.03 * 10 ⁵, 1.89 * 10 ⁵, 1.40 * 10 ⁵The total RNA of copies/5ng, be respectively 1/12.1,1/4.4,1/4.2,1/3.3,1/2.8,1/3.9,1/2.1,1/3.1,1/3.7,1/2.9,1/3.2 of transfection si control cells, the propagation of virus in cells infected that shown siRNA molecules in inhibiting that the Pestivirus suis of transfered cell is special.Reclaim Real-time PCR product, carry out sequencing, the result shows that the Real-time pcr amplified fragment is a Pestivirus suis Shimen strain NS5B gene fragment, illustrates that detected result has the specificity of height.

Table 3 is measured the half cell infection amount (TCID of virus in the cell ₅₀) the increase and decrease situation

The siRNA molecule	Infect the TCID of back 60h 50	With contrast ratio
			The si contrast	1×10 4.25	-
siN1	1×10 2.58	1/46.8
			siN2	1×10 3.08	1/31.6
siS2	1×10 3.11	1/13.8
			siS3	1×10 3.20	1/11.2
siS6	1×10 2.91	1/21.9
			siS7	1×10 3.26	1/9.8
siS8	1×10 3.13	1/13.2
			siS10	1×10 2.97	1/19.1
siS11	1×10 3.14	1/12.9

siS12	1×10 2.99	1/18.2
			si5B1	1×10 2.83	1/56.2

The result shows: after the cell infection Pestivirus suis of transfection contrast si contrast, virus can be bred in cell effectively, demonstrates higher infection titer, infects the TCID of back 60h ₅₀Measurement result is 1 * 10 ^4.25, with 60h behind the non-transfected cells infected pigs pestivirus quite, show that transfection reagent and transfection process pair cell support that the multiplication capacity of Pestivirus suis is effectively less, and after the cell infection Pestivirus suis of transfection Pestivirus suis siRNA molecule, TCID ₅₀Lower, 60h behind the virus infection, the TCID of siN1, siN2, si5B1, siS2, siS3, siS6, siS7, siS8, siS10, siS11, siS12 transfectional cell ₅₀Compare its TCID with the infection titer of si contrast ₅₀Be respectively 1/46.8,1/31.6,1/56.2,1/13.8,1/11.2,1/21.9,1/9.8,1/13.2,1/19.1,1/12.9,1/18.2 times, show that siN1, siN2 and si5B1 siN1, siN2, si5B1, siS2, siS3, siS6, siS7, siS8, siS10, siS11, siS12 have suppressed the propagation of Pestivirus suis effectively.

Example 2.

The shRNA that expresses suppresses Pestivirus suis propagation:

With containing 10% calf serum, not containing antibiotic MEM nutritive medium and go down to posterity the PK-15 cell in 24 porocyte culture plates, every hole 5.0 * 10 ⁴Cell/500 μ L, 37 ℃, 5%CO ₂Cultivate 20-24h in the incubator, make cell attachment growth full scale reach 90-95%, use Lipofectamine ^TM2000 (Invitrogen) carry out transfection, and (every porocyte) method is:

1. get among the MEM that 0.8 μ g plasmid DNA adds 50 μ L serum-free antibiotic-frees mixing.

2. get 2.0 μ L Lipofectamine ^TM2000 add among the MEM of 50 μ L serum-free antibiotic-frees, and mixing leaves standstill 5min in room temperature gently.

3. the Lipofectamine that the plasmid DNA of dilution is added dilution ^TMIn 2000, leave standstill 20min in room temperature behind the mixing gently, to form DNA-Lipofectamine ^TM2000 complex bodys.

4. with the DNA-Lipofectamine that forms ^TM2000 join above-mentioned preparation in, mixing places 37 ℃ of 5%CO gently ₂Transfection 4-6h in the incubator uses instead and contains 2% calf serum and antibiotic MEM, 37 ℃ of 5%CO ₂Cultivate standby in the incubator.

5. antibiotics resistance screening

With transfection the cell of shRNA expression plasmid, by optimizing the passage cell full scale of determining, with the MEM that contains 10% serum go down to posterity in 6 porocyte culture plates (the PK-15 cell of the untransfected of the identical full scale that goes down to posterity simultaneously is as the normal control cell of pressurization), after cultivating 24h, use instead and contain the corresponding microbiotic (G-418 or hygromicin B) that kills cell concn, the MEM that contains 2% serum, the cell that does not change plasmid over to is killed in 37 ℃ of cultivations.Use instead every 3d therebetween and contain antibiotic fresh nutrient solution, dead fully until control cells, the cell of transfection plasmid grows cell colony.Use instead and contain the 50% corresponding microbiotic that kills cell concn and continue to cultivate transfectional cell, after cell colony covered with culture hole, enlarged culturing screening gained cell was standby.

6. the shRNA that expresses suppresses the effect detection of Pestivirus suis: the transfectional cell that antibiotics resistance is arranged that screening obtains, go down to posterity in 96 porocyte culture plates with the MEM of antibiotic-free (G-418 or hygromicin B), behind the cultivation 20-24h, with 1 * 10 ²TCID ₅₀Pestivirus suis Shimen strain infect, cultivate 72h behind the virus infection, with the half cell infection amount (TCID of indirect immunofluorescence, Real-time PCR, virus ₅₀) method such as mensuration detects the propagation of Pestivirus suis in transfectional cell, measures the restraining effect of siRNA to viral gene expression in the swine fever virus infection breeding.

Table 4 indirect immunofluorescence assay detects the infected situation of cell

Express shRNA molecule plasmid	The indirect immunofluorescence result
		The sh contrast	Cell 100% is infected
shN1	Little fourth 10% cell is infected
		shN2	Infected less than 10% cell
shS6	Infected less than 10% cell
		shS7	Infected less than 10% cell
shS8	The 30%-60% cell is infected
		shS10	The 30%-60% cell is infected
shS11	Infected less than 10% cell
		shS2	Infected less than 10% cell
shS3	The 30%-60% cell is infected
		sh5B1	Infected less than 10% cell
shS12	The 30%-60% cell is infected

Indirect immunofluorescence assay detects the infected situation result of cell and shows, the shRNA molecules in inhibiting of design virus infected cell.

Table 5Real-time PCR method detects geneome RNA copy number measurement result

Express shRNA molecule plasmid	The geneome RNA number	Inhibiting rate	Suppress multiple
				The sh contrast	5.64×10 5	-	-
shN1	8.91×10 3	98.4	62.3
				shN2	1.28×10 4	97.0	43.1
shS6	1.65×10 4	97.1	33.2
				shS7	2.18×10 4	96.3	24.9
shS8	3.40×10 4	94.0	15.6

shS10	5.66×10 3	99.0	98.6
				shS11	8.49×10 3	98.5	65.1
shS2	7.54×10 3	98.7	73.8
				shS3	1.29×10 4	97.7	42.7
Sh5B1	7.19×10 3	98.7	77.4
				shS12	3.99×10 4	92.9	13.1

Annotate: inhibiting rate=(contrast molecule number-shRNA sample molecule number)/contrast molecule number * 100%, calculate each shRNA and suppress the efficient that viral genome is duplicated, suppress the value-1 of multiple in the table for contrast/shRNA.The Real-time PCR method detects geneome RNA copy number measurement result and shows that the genome duplication of Pestivirus suis in transfectional cell has been subjected to suppressing efficiently.

Different time TCID behind the table 6 stable transfected cells infected pigs pestivirus ₅₀Measurement result

Express shRNA molecule plasmid	12h	24h	36h	48h	60h	72h	96h	120h
									The Sh contrast	1×10 1.92	1×10 3.67	1×10 1.33	1×10 4.58	1×10 4.58	1×10 4.12	1×10 4.33	1×10 4.08
shN1	1×10 1.17	1×10 1.67	1×10 1.83	1×10 2.58	1×10 2.83	1×10 3.33	1×10 3.58	1×10 3.17
									shS6	1×10 1.67	1×10 2.12	1×10 3.08	1×10 3.42	1×10 3.6	1×10 3.92	1×10 3.83	1×10 3.83
shS10	1×10 1.33	1×10 1.92	1×10 2.42	1×10 2.83	1×10 3.17	1×10 3.58	1×10 3.83	1×10 3.12
									shS11	1×10 1.58	1×10 2.58	1×10 3.17	1×10 3.12	1×10 3.58	1×10 3.67	1×10 3.92	1×10 3.58
shS2	1×10 1.17	1×10 1.83	1×10 2.12	1×10 2.92	1×10 3.33	1×10 3.67	1×10 3.83	1×10 3.67
									shS3	1×10 1.67	1×10 2.12	1×10 2.92	1×10 3.17	1×10 3.58	1×10 3.92	1×10 3.9	1×10 3.83
Sh5B1	1×10 1.08	1×10 1.83	1×10 2.58	1×10 3.08	1×10 3.33	1×10 3.58	1×10 3.67	1×10 3.42
									shS12	1×10 1.67	1×10 2.38	1×10 2.92	1×10 3.17	1×10 3.58	1×10 3.67	1×10 3.66	1×10 3.58
shN2	1×10 1.18	1×10 2.34	1×10 2.85	1×10 3.31	1×10 3.54	1×10 3.71	1×10 3.80	1×10 3.65
									shS7	1×10 1.21	1×10 2.56	1×10 3.12	1×10 3.68	1×10 3.73	1×10 3.69	1×10 3.78	1×10 3.72
shS8	1×10 1.30	1×10 2.10	1×10 2.04	1×10 3.15	1×10 3.58.	1×10 3.48	1×10 3.68	1×10 3.65

Detected result shows: the cell of transfection sh contrast, behind swine fever virus infection, its infection titer increases rapidly, 48-60h peaks after infection, the variation characteristics of its infection titer when Pestivirus suis is bred have been embodied in the PK-15 cell, and compare with control cells, the cell of the shRNA expression plasmid that the transfection Pestivirus suis is special, the infection titer of Pestivirus suis gathers way slowly, and the infection titer than control cells is much lower, when the infection titer in control cells peaks (48-60h), the infection titer of the cell of the shRNA expression plasmid that the transfection Pestivirus suis is special still remains on lower level, and present comparatively level and smooth rising tendency, the special shRNA of Pestivirus suis that shows plasmid expression makes the assembling process of sophisticated infective virus particle of Pestivirus suis be subjected to inhibition, and restraining effect can continue the long time, even in the highest time of infection titer (72-96h), its infection titer is still compared according to low order of magnitude after the cell infection virus of the shRNA expression plasmid that the transfection Pestivirus suis is special.

Detected result shows, 60h after infection presses sh contrast/shRNA-1 and calculates the inhibition multiple, and then shN1, shS6, shS10, shS11, shS2, shS3, sh5B1, shS12, shN2, shS7, shS8 suppress Pestivirus suis TCID ₅₀Multiple be respectively 56.3,8.2,25.9,10.1,17.9,5.6,17.9,5.6,11.0,6.0,9.0 times, even at shRNA transfectional cell TCID ₅₀Higher 96h still has 4-10 inhibition efficient doubly to virus.

Example 3

The experimentation on animals level

1. buy 50 Japanese white big ear rabbits, be numbered, breeding observing 7d measures rabbit arm's length basis body temperature and measures twice every day, and the rabbit of numbering is carried out random packet with SPSS software, and 8 every group, grouping and injection see Table 3.

The grouping of table 3 experimental rabbit

Group	SiRNA (μ g)/only	Plasmid (μ g)/only
			Si contrast siN1 siN2 sh contrast shN1 shN2	300 300 300 - - -	- - - 500 500 500

After the experimental rabbit grouping, by the injected dose shown in the last table, the siRNA and the shRNA expression plasmid of acquisition transcribed in injection respectively, wherein plasmid 24h injection before attacking poison, and the injection site is that neck is subcutaneous, siRNA and attack poison and inject simultaneously is intravenous injection.

2. HCLV attacks poison and body temperature mensuration: the rabbit of injection shRNA expression plasmid and siRNA, and by intravenous injection 50 * ID ₅₀The hog cholera lapinised virus strain, every 12h measures 2 body temperature in the 24h of injection back, every interval 6h measures rabbit body temperature behind the 24h, returns to the normal body temperature value up to rabbit body temperature.

Table 7 respectively organize rabbit mean body temperature (℃)

Time	The si contrast	siN1	siN2	The sh contrast	shN1	shN2
							Basal body temperature 0 12 24 30 36 42 48 54 60 66 72	39.2 39.3 39.2 39.3 39.3 39.4 39.8 39.9 40.1 40.4 40.3 40.5 39.9 39.4	39.2 39.4 39.3 39.4 39.2 39.4 39.3 39.6 39.5 39.8 39.9 39.9 39.5 39.4	39.2 39.4 39.3 39.3 39.3 39.2 39.5 39.3 39.5 39.5 39.9 40.0 40.2 39.6	39.3 39.3 39.2 39.2 39.4 39.5 39.6 39.9 39.8 40.2 40.5 40.4 39.8 39.4	39.2 39.2 39.3 39.2 39.4 39.3 39.5 39.5 39.4 39.3 39.7 40.2 39.9 39.4	39.4 39.2 39.3 39.3 39.5 39.4 39.5 39.3 39.5 39.9 40.4 39.9 40.1 39.6

It is as shown in the table, in the siRNA of in-vitro transcription experimental group: in si contrast, siN1 and three groups of siN2,0.5 ℃ the time that the rabbit of the injection si control group of transcribing is heated up first above basal body temperature is at 30h, animal continues to occur high heat afterwards, and can continue 6 temperature time (36h) to attacking poison back 60h, and obviously postponing than control group of fever time appears in experimental group, the time of siN1 group is being attacked poison back 48h, fever time than control group has postponed 12h, and heating has only 3 warm time; The heating time of occurrence of siN2 injection group has postponed 18h attacking poison back 54h than control group, and is same, and its fever time has only continued 18h (3 temperature are inferior), and the mean body temperature of the pyrogenic stage of two groups all is lower than control group.In three experimental group of injection shRNA expression plasmid, the time of the fever in rabbits of injection sh contrast expression plasmid appears at 36h, time length is 36h, and the fever time of experimental group shN1 has postponed 18h at 54h than control group, and the time length is a 18h institute, shN2 injection group the time of heating occurs at 48h, different with other three experimental group is that the heating continuing time of shN2 group is long slightly, is 24h (4 temperature are inferior).

Experimental result from table is found out, the experimental group of the special RNA interfering of injection Pestivirus suis all shows fever time and postpones, the heating medial temperature is more lower slightly than control group, and the characteristics that heating continuing time shortens greatly than control group, show that the special RNA interfering of Pestivirus suis has postponed the hog cholera lapinised virus strain in the intravital propagation of rabbit, postponed making rabbit the required virus quantity of high fever reaction occur.As can be seen, the body temperature rise speed of experimental group is obviously slow than control group in the rabbit body temperature changing trend diagram from Fig. 3, the weak point of the hot time ratio control group of height occurs continuing, and temperature is low than control group.

3. suppressing virus multiplication measures: return to the rabbit of normal body temperature, catch and kill in 6h, aseptic collection rabbit spleen and mesenteric lymph nodes are with the half cell infection amount (TCID of Real-time PCR, virus ₅₀) method such as mensuration detects the propagation of Pestivirus suis in the rabbit body, measures the restraining effect of siRNA molecule to viral gene expression in the swine fever virus infection breeding.

With 50 * ID ₅₀Pestivirus suis rabbitization low virulent strain attack the body temperature that rabbit is measured in poison back, extract the total RNA of rabbit spleen, detect viral genome propagation situation with Real-time PCR, the result shows and sees Table 8.

Table 8 experimental rabbit geneome RNA measurement result

Group	Genome copy number ratio (experimental group/control group)
									The si contrast	-	-	-	-	-	-	-	-
siN1	1/13.2	1/2.77	1/4.83	1/8.42	1/9.13	1/1.62	1/4.16	1/17.83
									siN2	1/2.37	1/2.93	1/10.07	1/6.2	1/2.43	1/3.02	1/6.04	1/37.57
The sh contrast	1/1.25	1/1.34	1/1.40	1/1.10	1/1.07	1/2.16	1/1.12	1/1.07
									shN1	1/3.11	1/1.04	1/7.47	i/30.26	1/6.15	1/1.46	1/6.45	1/3.91
shN2	1/6.54	1/3.21	1/32.37	1/5.02	1/4.72	1/2.04	1.14/1	1/1.97

The genome quantity of control group si contrast and sh contrast fluctuates on higher level, and the genome quantity in the experimental group is then lower, proves that virus multiplication is suppressed.

Get the spleen of respectively organizing experimental rabbit of collection, balanced mix is ground the back and is measured their infection titer, sees Table 9.

Table 9 rabbit spleen inner virus infection titer detected result

Group	Rabbit spleen inner virus infection titer
			ID 50	Experimental group/contrast
	The si contrast	1×10 3.83			-
siN1			1×10 3.08	1/5.62
	siN2	1×10 3.33			1/2.15
The sh contrast			1×10 3.92	-
	shN1	1×10 3.33			1/3.87
shN2			1×10 3.17	1/5.58

The result shows, the si contrast, and siN1, siN2, the sh contrast, N1, the ID of pairing HCLV is organized in the N2 injection ₅₀Be respectively 1 * 10 ^3.83, 1 * 10 ^3.08, 1 * 10 ^3.33, 1 * 10 ^3.92, 1 * 10 ^3.33, 1 * 10 ^3.17, the ID of control group si contrast ₅₀Be respectively siN1 and siN2 5.62 times and 2.15 times, the ID of sh contrast ₅₀Be respectively N1 and N2 3.87 times and 5.58 times.Measurement result shows that siRNA and shRNA expression plasmid that injection is transcribed have all suppressed the hog cholera lapinised virus strain effectively in the intravital propagation of rabbit.

Positively effect of the present invention is: use the biotechnological formulation of RNA interference method production for treating swine fever virus infection, obtained the disturbance RNA molecule of efficient, special inhibition Pestivirus suis, be applicable to suitability for industrialized production.The present invention also provides the biotechnological formulation that utilizes this side to produce, and up to 92.9-99.0%, can obviously stop the infection of CSFV to the inhibition efficient of CSFV in responsive animal body, makes animal avoid sickness rate and death.

Description of drawings

Fig. 1 is the synthetic siRNA molecular schematic diagram of in-vitro transcription of the present invention.

Fig. 2 is the synoptic diagram of shRNA molecule of the present invention.

Fig. 3 rabbit is attacked the temperature curve behind the poison.

Embodiment

Embodiment 1 siRNA design

Analyze the Pestivirus suis genome sequence of having delivered among the GenBank, select zone conservative in the genome, according to the requirement of RNAi technology, searching has AA-N ₁₉The 21nt sequence of feature, carry out BlastN and secondary structure analysis, according to the BlastN analytical results, with physical and chemical parameters such as their (G+C) % of VNTI3.0 software analysis, Tm value, inner hairpin ring structures, select the lower sequence of (G+C) %, designed 11 siRNA molecule interference sequences at each gene of Pestivirus suis.

Designed sequence and the position in the Pestivirus suis genome thereof are as shown in table 10:

Table 10siRNA molecule interference sequence and position thereof

At gene	The siRNA sequence	The position *	Name
				Npro	AAGGATAGGTAGGGTGACAGG	721-741	N1
Npro	AAGAACCCTGAAGTGGATTAG	820-840	N2
				C	AAGTGGAAGTAAAGAGAAGAA	892-912	S6
Erns	AAGAATAAACCACCAGAATCT	1082-1102	S7
				E1	AAGGTGGCTTTGTTATTTGAA	2255-2275	S8
NS2-3	AAGATGACTAATGGACTTAGT	3923-3943	S10
				NS4A	AAGAGGCATATACCAGTAGTCA	7253-7274	S11
NS4B	AAGCTGGAATTACAATAATCT	7837-7857	S2
				NS5A	AAGATGAAGGCAGTTAAGAAT	8606-8626	S3
NS5B	AAGAATGAGAAGAGGGACGTC	10700-10720	5B1
				3’UTR	AAGTTACCACACTACACTCAT	12205-12225	S12

*: the position relatively with reference to strain shimen, GenBank accession number: AF092448.

Embodiment 2 external synthetic siRNA:

Press T7 RiboMAX ^TMThe dna sequence dna and the T7 RNA polymerase binding sequence of 11 selected segmental siRNA molecules among the synthetic embodiment 1 of Express RNAi System (Promega) specification sheets design are referring to accompanying drawing 1; Synthesize respectively and transcribe the positive-sense strand of each siRNA molecule and the template DNA of antisense strand, sequence is as follows:

T7 RNA polymerase binding sequence: GGATCCTAATACGACTCACTATA.

5B1 positive-sense strand template sequence: AAGACGTCCCTCTTCTCATTCTATAGTGAGTCGTATTAGGATCC

The template sequence of 5B1 antisense strand: AAGAATGAGAAGAGGGACGTCTATAGTGAGTCGTATTAGGATCC

N1 positive-sense strand template sequence: AACCTGTCACCCTACCTATCCTATAGTGAGTCGTATTAGGATCC

The template sequence of N1 antisense strand: AAGGATAGGTAGGGTGACAGGTATAGTGAGTCGTATTAGGATCC

N2 positive-sense strand template sequence: AACTAATCCACTTCAGGGTTCTATAGTGAGTCGTATTAGGATCC

The template sequence of N2 antisense strand: AAGAACCCTGAAGTGGATTAGTATAGTGAGTCGTATTAGGATCC

S6 positive-sense strand template sequence: AATTCTTCTCTTTACTTCCACTATAGTGAGTCGTATTAGGATCC

The template sequence of S6 antisense strand: AAGTGGAAGTAAAGAGAAGAATATAGTGAGTCGTATTAGGATCC

S7 positive-sense strand template sequence: AAAGATTCTGGTGGTTTATTCTATAGTGAGTCGTATTAGGATCC

The template sequence of S7 antisense strand: AAGAATAAACCACCAGAATCTTATAGTGAGTCGTATTAGGATCC

S8 positive-sense strand template sequence: AATTCAAATAACAAAGCCACCTATAGTGAGTCGTATTAGGATCC

The template sequence of S8 antisense strand: AAGGTGGCTTTGTTATTTGAATATAGTGAGTCGTATTAGGATCC

S10 positive-sense strand template sequence: AAACTAAGTCCATTAGTCATCTATAGTGAGTCGTATTAGGATCC

The template sequence of S10 antisense strand: AAGATGACTAATGGACTTAGTTATAGTGAGTCGTATTAGGATCC

S11 positive-sense strand template sequence: AATGACTACTGGTATATGCCTCTATAGTGAGTCGTATTAGGATCC

The template sequence of S11 antisense strand: AAGAGGCATATACCAGTAGTCATATAGTGAGTCGTATTAGGATCC

S12 positive-sense strand template sequence: AAATGAGTGTAGTGTGGTAACTATAGTGAGTCGTATTAGGATCC

The template sequence of S12 antisense strand: AAGTTACCACACTACACTCATTATAGTGAGTCGTATTAGGATCC

S2 positive-sense strand template sequence: AAAGATTATTGTAATTCCAGCTATAGTGAGTCGTATTAGGATCC

The template sequence of S2 antisense strand: AAGCTGGAATTACAATAATCTTATAGTGAGTCGTATTAGGATCC

S3 positive-sense strand template sequence: AAATTCTTAACTGCCTTCATCTATAGTGAGTCGTATTAGGATCC

The template sequence of S3 antisense strand: AAGATGAAGGCAGTTAAGAATTATAGTGAGTCGTATTAGGATCC

The positive-sense strand template sequence of contrast: AACCTATCCATCCCACTGTCCTATAGTGAGTCGTATTAGGATCC

The template sequence of the antisense strand of contrast: AAGGACAGTGGGATGGATAGGTATAGTGAGTCGTATTAGGATCC

Concrete steps are:

It is 100pmol/L that above-mentioned synthetic DNA is diluted respectively with the water of no DNA enzyme, no RNA enzyme, synthetic respectively by the following step 11 siRNA molecules.

With PCR method they are synthesized double-stranded DNA respectively, no DNA enzyme in cleaning, the T7 RNA polymerase binding sequence DNA 5.0 μ L that add dilution in the PCR pipe of no RNA enzyme, add respectively above-mentioned 11 be used for being transcribed into the siRNA molecule positive-sense strand and antisence strand dna 5.0 μ L, 10 * pfu DNA polymerasebuffer, 5.0 μ L, 10mmol/L dNTP 1.0 μ L, water 33.8 μ L, place 95 ℃ of sex change 2min of PCR instrument, add pfu DNA polymerase 0.2 μ L (1U), again in 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 10s, after 5 circulations, extend 7min after 72 ℃.After reaction finishes, adding is handled and autoclaved 3mol/L sodium-acetate (pH 5.2) 5.0 μ L through DEPC, the dehydrated alcohol that adds 2.5 times of volumes then, place 10min, the centrifugal 10min of 12000r/min, abandoning supernatant in-20 ℃ behind the mixing, precipitate with 70% washing with alcohol, be deposited in drying at room temperature 10min, add the abundant dissolution precipitation of water 50 μ L of no DNA enzyme, no RNA enzyme, the polyacrylamide gel electrophoresis checks that template DNA prepares the result.Dubbing method is pressed T7RiboMAX ^TMExpress RNAi System specification sheets carries out body: add the template DNA 4.0 μ L of above-mentioned preparation successively in the PCR pipe of the no DNA enzyme of cleaning, no RNA enzyme, RiboMAX ^TMExpress T7 2 * Buffer 10.0 μ L, Nuclease-Free Water 4.0 μ L, Enzyme Mix, T7Express 2.0 μ L place 37 ℃ of reaction 1h then; The DNA enzyme 1.0 μ L (1U) that add no RNA enzyme, 37 ℃ of 30min; Mix corresponding siRNA justice and antisense strand transcript, 70 ℃ of insulation 10min slowly are cooled to room temperature; The 3mol/L sodium-acetate (pH 5.2) that adds 1/10 volume, add isopyknic Virahol, ice bath 5min behind the mixing, the centrifugal 10min of 13000r/min, the careful suction removed supernatant liquor, 500 μ L, 70% washing with alcohol precipitation, be deposited in drying at room temperature 10-15min, add the abundant dissolution precipitation of 100 μ L water, electrophoretic examinations, be packed as 0.3 μ g/ μ L after measuring content ,-80 ℃ of preservations are standby.Obtain external synthetic siRNA swine fever treatment biotechnological formulation, its base sequence sees Table 11.

The external synthetic siRNA of table 11 swine fever treatment biotechnological formulation and contrast siRNA molecule

The siRNA title	The biological products base sequence of external synthetic siRNA swine fever treatment
		siN1	GGAUAGGUAGGGUGACAGGUU UUCCUAUCCAUCCCACUGUCC
siN2	GAACCCUGAAGUGGAUUAGUU UUCUUGGGACUUCACCUAAUC
		siS6	GUGGAAGUAAAGAGAAGAAUU UUCACCUUCAUUUCUCUUCUU
siS7	GAAUAAACCACCAGAAUCUUU UUCUUAUUUGGUGGUCUUAGA
		siS8	GGUGGCUUUGUUAUUUGAAUU UUCCACCGAAACAAUAAACUU
siS10	GAUGACUAAUGGACUUAGUUU UUCUACUGAUUACCUGAAUCA
		siS11	GAGGCAUAUACCAGUAGUCAUU UUCUCCGUAUAUGGUCAUCAGU

siS2	GCUGGAAUUACAAUAAUCUUU UUCGACCUUAAUGUUAUUAGA
		siS3	GAUGAAGGCAGUUAAGAAUUU UUCUACUUCCGUCAAUUCUUA
si5B1	GAAUGAGAAGAGGGACGUCUU UUCUUACUCUUCUCCCUGCAG
		siS12	GUUACCACACUACACUCAUUU UUCAAUGGUGUGAUGUGAGUA
The si contrast	GGACAGUGGGAUGGAUAGGUU UUCCUGUCACCCUACCUAUCC

The method of design of embodiment 3 plasmid expression shRNA molecules:

According to the requirement of pSilencer3.1H1Hygro (Ambion) shRNA carrier design, be designed for the dna sequence dna of expressing shRNA, layout strategy sees Table 12.In order to improve the efficient that the correct annealing of sequence becomes double chain DNA molecule, be designed to strategy with archaeal dna polymerase synthetic PCR, the dna sequence dna of expressing purpose shRNA sequence is split as the sequence of two sections part complementary pairings, use the VNTI3.0 assistant analysis then, making the complementary pairing base between two sections sequences of fractionation is 21-23bp, be less than 10bp and respectively split sequence self pairing base, to improve the efficient of the synthetic double-stranded template DNA of PCR.

Design synthetic each shRNA template DNA sequence such as table 12:

Table 12 generates the template DNA sequence of shRNA molecule

Name	The template DNA sequence
		N1	GACGGATCCGGATAGGTAGGGTGACAGGTTCAAGAGACCTGTC
TGCAAGCTTTTCCAAAAAAGGATAGGTAGGGTGACAGGTCTCTTGAACCTGT
	N2		GACGGATCCGAACCCTGAAGTGGATTAGTTCAAGAGACTAATC
TGCAAGCTTTTCCAAAAAAGAACCCTGAAGTGGATTAGTCTCTTGAACTAATC
		S6	GGAGGATCCCGTGGAAGTAAAGAGAAGAATTCAAGAGATTCTTC
CCGAAGCTTTTCCAAAAAAGTGGAAGTAAAGAGAAGAATCTCTTGAATTCTTCTC
	S7		GGAGGATCCCGAATAAACCACCAGAATCTTTCAAGAGAAGATTC
CCGAAGCTTTTCCAAAAAAGAATAAACCACCAGAATCTTCTCTTGAAAGATTCTG
		S8	GGAGGATCCCGGTGGCTTTGTTATTTGAATTCAAGAGATTCAA
CCGAAGCTTTTCCAAAAAAGGTGGCTTTGTTATTTGAATCTCTTGAATTCAAAT
	S10		GGAGGATCCCGATGACTAATGGACTTAGTTTCAAGAGAACTAAG
CCGAAGCTTTTCCAAAAAAGATGACTAATGGACTTAGTTCTCTTGAAACTAAGTC
		S11	GGAGGATCCCGAGGCATATACCAGTAGTCATTCAAGAGATGACTAC
CCGAAGCTTTTCCAAAAAAGAGGCATATACCAGTAGTCATCTCTTGAATGACTAC
	S2		GGAGGATCCCGCTGGAATTACAATAATCTTTCAAGAGAAGATTATTG
CCGAAGCTTTTCCAAAAAAGCTGGAATTACAATAATCTTCTCTTGAAAGATT
		S3	GGAGGATCCCGATGAAGGCAGTTAAGAATTTCAAGAGAATTC

	CCGAAGCTTTTCCAAAAAAGATGAAGGCAGTTAAGAATTCTCTTGAAATTCTTAAC
		5B1	GACGGATCCGAATGAGAAGAGGGACGTCTTCAAGAGAGACGTCC
TCGAAGCTTTTCCAAAAAAGAATGAGAAGAGGGACGTCTCTCTTGAAGACG
	S12		GGAGGATCCCGTTACCACACTACACTCATTTCAAGAGAATGAGTG
CCGAAGCTTTTCCAAAAAAGTTACCACACTACACTCATTCTCTTGAAATGAGTG
		Contrast	GACGGATCCGGACAGTGGGATGGATAGGTTCAAGAGACCTATC
GGCAAGCTTTTCCAAAAAAGGACAGTGGGATGGATAGGTCTCTTGAACCTATCC

Concrete steps are as follows:

Synthetic respectively by the following step 11 plasmids that generate the shRNA molecule, with synthetic DNA dilution is 100 μ mol/L, with PCR method they are synthesized double-stranded DNA respectively, method is: the DNA 5.0 μ L that get dilution, add 10 * pfu DNA polymerase buffer, 5.0 μ L, 10mmol/L dNTP 1.0 μ L, water 33.8 μ L, place 95 ℃ of sex change 2min of PCR instrument, add pfu DNA polymerase 0.2 μ L (1U), again in 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 10s after 5 circulations, extend 7min after 72 ℃.After reaction finishes, add 3mol/L sodium-acetate (pH 5.2) 5.0 μ L, the dehydrated alcohol that adds 2.5 times of volumes is then placed 10min, the centrifugal 10min of 12000r/min in-20 ℃ behind the mixing, abandoning supernatant, with 1000 μ L, 70% washing with alcohol precipitation, abandoning supernatant is deposited in drying at room temperature 10min, add the abundant dissolution precipitation of 50 μ L water ,-80 ℃ of preservations are standby.The DNA that expresses shRNA with BamHI and HindIII double digestion inserts fragment, enzyme is cut digestion and is finished the back in 70 ℃ of insulation 15min deactivation restriction endonucleases, phenol/chloroform extracting, respectively they are cloned into the BamHI and the HindIII site of pSilencer3.1H1 Hygro carrier behind the ethanol sedimentation, transformed into escherichia coli, carry out PCR Rapid identification with 31H1FP (GTGCTGCAAGGCGATTAAGTTGG) and 31H1RP (GCGGATAACAATTTCACACAGG) for primer to transforming bacterium colony, the PCR positive colony is extracted plasmid in a small amount, determine the segmental exactness of insertion in the recombinant plasmid by checking order.

The shRNA molecular energy of the plasmid expression that obtains suppresses Pestivirus suis propagation, and the pSilencer3.1H1 Hygro carrier of expressing the shRNA molecule inserts sequence and sees Table 13 between BamHI and HindIII restriction enzyme site.

The pSilencer3.1H1Hygro carrier that table 13 is expressed the shRNA molecule inserts sequence between BamHI and HindIII restriction enzyme site

The shRNA title	Sequence base sequence between the BamHI of insertion carrier and HindIII site
		shN1	GATCCGGATAGGTAGGGTGACAGGTTCAAGAGACCTGTCACCCTACCTATCCTTTTTTGGAAA GCCTATCCATCCCACTGTCCAAGTTCTCTGGACAGTGGGATGGATAGGAAAAAACCTTTTCGA
shN2	GATCCGAACCCTGAAGTGGATTAGTTCAAGAGACTAATCCACTTCAGGGTTCTTTTTTGGAAA GCTTGGGACTTCACCTAATCAAGTTCTCTGATTAGGTGAAGTCCCAAGAAAAAACCTTTTCGA
		shS6	GATCCCGTGGAAGTAAAGAGAAGAATTCAAGAGATTCTTCTCTTTACTTCCACTTTTTTGGAAA GGCACCTTCATTTCTCTTCTTAAGTTCTCTAAGAAGAGAAATGAAGGTGAAAAAACCTTTTCGA

shS7	GATCCCGAATAAACCACCAGAATCTTTCAAGAGAAGATTCTGGTGGTTTATTCTTTTTTGGAAA GGCTTATTTGGTGGTCTTAGAAAGTTCTCTTCTAAGACCACCAAATAAGAAAAAACCTTTTCGA
		shS8	GATCCCGGTGGCTTTGTTATTTGAATTCAAGAGATTCAAATAACAAAGCCACCTTTTTTGGAAA GGCCACCGAAACAATAAACTTAAGTTCTCTAAGTTTATTGTTTCGGTGGAAAAAACCTTTTCGA
shS10	GATCCCGATGACTAATGGACTTAGTTTCAAGAGAACTAAGTCCATTAGTCATCTTTTTTGGAAA GGCTACTGATTACCTGAATCAAAGTTCTCTTGATTCAGGTAATCAGTAGAAAAAACCTTTTCGA
		shS11	GATCCCGAGGCATATACCAGTAGTCATTCAAGAGATGACTACTGGTATATGCCTCTTTTTTGGAAA GGCTCCGTATATGGTCATCAGTAAGTTCTCTACTGATGACCATATACGGAGAAAAAACCTTTTCGA
shS2	GATCCCGCTGGAATTACAATAATCTTTCAAGAGAAGATTATTGTAATTCCAGCTTTTTTGGAAA GGCGACCTTAATGTTATTAGAAAGTTCTCTTCTAATAACATTAAGGTCGAAAAAACCTTTTCGA
		shS3	GATCCCGATGAAGGCAGTTAAGAATTTCAAGAGAATTCTTAACTGCCTTCATCTTTTTTGGAAA GGCTACTTCCGTCAATTCTTAAAGTTCTCTTAAGAATTGACGGAAGTAGAAAAAACCTTTTCGA
sh5B1	GATCCGAATGAGAAGAGGGACGTCTTCAAGAGAGACGTCCCTCTTCTCATTCTTTTTTGGAAA GCTTACTCTTCTCCCTGCAGAAGTTCTCTCTGCAGGGAGAAGAGTAAGAAAAAACCTTTTCGA
		shS12	GATCCCGTTACCACACTACACTCATTTCAAGAGAATGAGTGTAGTGTGGTAACTTTTTTGGAAA GGCAATGGTGTGATGTGAGTAAAGTTCTCTTACTCACATCACACCATTGAAAAAACCTTTTCGA
The sh contrast	GATCCGGACAGTGGGATGGATAGGTTCAAGAGACCTATCCATCCCACTGTCCTTTTTTGGAAA GCCTGTCACCCTACCTATCCAAGTTCTCTGGATAGGTAGGGTGACAGGAAAAAACCTTTTCGA

Claims (3)

1, sequence on the following Pestivirus suis genome, utilize the synthetic RAN disturbing molecule of RNA perturbation technique design after, thereby these rnai molecules can special, efficiently suppress Pestivirus suis propagation swine fever virus infection is had obvious therapeutic action, it is characterized in that:

AAGGATAGGTAGGGTGACAGG(721-741)；

AAGAACCCTGAAGTGGATTAG(820-840)；

AAGTGGAAGTAAAGAGAAGAA(892-912)；

AAGAATAAACCACCAGAATCT(1082-1102)；

AAGGTGGCTTTGTTATTTGAA(2255-2275)；

AAGATGACTAATGGACTTAGT(3923-3943)；

AAGAGGCATATACCAGTAGTCA(7253-7274)；

AAGCTGGAATTACAATAATCT(7837-7857)；

AAGATGAAGGCAGTTAAGAAT(8606-8626)；

AAGAATGAGAAGAGGGACGTC(10700-10720)；

AAGTTACCACACTACACTCAT(12205-12225)。

2, application rights requires the biotechnological formulation method that sequence is made on the 1 described Pestivirus suis genome, may further comprise the steps:

Positive-sense strand and the antisense strand template DNA and the T7 RNA polymerase binding sequence of the siRNA molecule of the synthetic described sequence of claim 1 utilize T7 RiboMAX ^TMThe siRNA molecule is synthesized in the system design of Express RNAiSystem (Promega) in-vitro transcription, obtains the siRNA molecule swine fever treatment biotechnological formulation that in-vitro transcription generates.

3, application rights requires the biotechnological formulation method that sequence is made on the 1 described Pestivirus suis genome, may further comprise the steps:

According to pSilencer3.1H1 Hygro (Ambion) shRNA carrier using method, the synthetic plasmid that can generate the shRNA molecule at sequence on the Pestivirus suis genome according to claim 1 obtains swine fever treatment biotechnological formulation.

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