CN102352359A - Anti-pig O type foot and mouth disease virus shRNA design and carrier construction method - Google Patents
Anti-pig O type foot and mouth disease virus shRNA design and carrier construction method Download PDFInfo
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Abstract
The invention relates to an anti-pig O type foot and mouth disease virus shRNA and carrier construction method, which is characterized in that: according to the O type foot and mouth disease virus genome sequence which is published by the GenBank, the protective sections of VP1 and 3D genes are selected, and a section of nucleic acid sequence of 19bp long is selected; and the nucleic acid sequence of 19bp is the specific target gene which only aims at the pig foot and mouth disease virus, does not have more similarity with the other genes, and the DNA sequence which corresponds to the hairpin-shaped shRNA is synthetized according to the characteristics of the carrier. The method adopts the synthesis method and utilizes the genetic engineering technology to construct the plasmid and screen the excellent interference sections, the plasmid has the capability of restraining the replication of the foot and mouth disease virus, and can obviously restrain the replication of the foot and mouth disease virus (FMDV) at the cell level; and the VP1 and 3D genes in FMDV gene open reading frames are selected according to the RNA interference technological principle as the target points to carry out RNA interference. When the virus RNA is replicated, firstly, a complementary strand is synthesized from the 3' end of the plus strand RNA under the 3D action; and then the negative strand is taken as the template to synthesize the progeny plus strand RNA. The nucleotide sequence and the amino acid sequence of 3Dpol have high conservative property.
Description
Technical field
The present invention relates to a kind of anti-pig O type hoof-and-mouth disease viral disease shRNA and construction of carrier; Particularly relate to a kind of genetic engineering technique that utilizes; Design through shRNA, synthetic and vector construction, attack process such as poison, RT-PCR and utilize the RNA perturbation technique to suppress pig O type foot and mouth disease virus and duplicate, belong to and suppress foot and mouth disease virus (FMDV) replica technique field.
Background technology
(Foot-and-mouth disease is by foot and mouth disease virus (Foot-and-mouth disease virus, a kind of serious harm that FMDV) causes acute, hot and height contagious disease artiodactylous FMD) to foot and mouth disease.This disease is widely current in countries in the world.This disease is rapid to propagate, sickness rate is high, financial loss is huge and become No.1 " economic sick " in the disease of domestic animals, and International Office of Epizootics (OIE) classifies it as first of the category-A livestock contagious disease, and China Ministry of Agriculture classifies it first disease of first kind animal epidemic as.Control to foot and mouth disease; Developing country is main with vaccine control mainly; But the development that in the immune prevention and control of this disease, has vaccine lag behind anti-strong, the deactivation of the variation, virulence of virus not thoroughly, the drawback of live virus escape production plant etc., this will ask for help explore the strategy and the means of new foot-and-mouth disease virus resistant.
Foot and mouth disease virus has A, O, C, Asia1 and 7 serotypes of South Africa 1,2,3 types, and different subtype has more than 70.In recent years, constantly occurred a lot of foot and mouth disease virus variants again in China different areas, this has brought very big difficulty for these sick prevention and control.To the control of foot and mouth disease, developed country takes to slaughter the measure of destruction mostly, and China mainly relies on vaccine immunization.FMD vaccine commonly used comprises inactivated vaccine and attenuated vaccine, and FMD inactivated vaccine immunogenicity is relatively poor, and spectrotype is narrower; Though attenuated vaccine has good immunogenicity; In the process of prevention and control FMD, play an important role, but exist unsafe factors such as virulence reversion, inactivation of virus are not thorough, live virus escape vaccine source mill to limit the application of this type of vaccine owing to foot and mouth disease.Simultaneously, because FMDV is a kind of RNA viruses serotype of height variability, and various do not exist cross-protection, prevents and control the popular more and more difficult that becomes of foot and mouth disease through immunization.
RNA disturbs (RNA interference; RNAi) be gene silent technology after a kind of the transcribing that is found in the earliest in the vegetable cell; It is meant the phenomenon of the efficient specificity degraded of high conservative during evolution, that brought out by double-stranded RNA, homologous mRNA; Also promptly goal gene can not be expressed through the degraded of goal gene being transcribed the back mRNA that produces; It is so-called gene silencing; Its principle is that this RNA is called small molecules interference RNA (being siRNA) through generation and said target mrna sequence specific complementary small molecules double-stranded RNA in importing or the cell.After siRNA produces, through chain among the siRNA and said target mrna sequence complementary pairing, again under the effect of a series of enzymes with the said target mrna degraded, thereby make said target mrna can not translate, express, make its phenotype silence.
After RNAi finds; The RNAi technology has been widely used in gene function, genetic development, has grown, signal transduction, field such as antitumor and antiviral; In antiviral research, fully demonstrate its huge application potential, be expected to become a kind of new tool of prevention and control viral blight.Therefore, utilize RNA perturbation technique construction of expression vector can suppress foot and mouth disease virus and duplicate, the transgenic animal that have foot-and-mouth disease virus resistant for next step cultivation provide powerful guarantee.
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Summary of the invention
The object of the present invention is to provide a kind of anti-pig O type hoof-and-mouth disease viral disease shRNA and construction of carrier; It adopts synthesis method and utilizes genetic engineering technique to make up plasmid and also outstanding interference fragment is screened; This plasmid has the ability that the inhibition foot and mouth disease virus is duplicated, and on cell levels, can obviously suppress duplicating of FMDV; We select the VP in the FMDV gene open reading frame according to RNA perturbation technique principle
1With the 3D gene for carrying out RNA interferential target site.VP wherein
1The coding region is the major antigen district, can produce neutralizing antibody by induced animal.3D is the RNA polymerase (3Dpol) that RNA relies on, the malicious related antigen (VIAA) of pretending illness again, catalysis viral RNA synthetic.At first be that positive chain RNA is under the 3D effect, from synthetic its complementary strand of its 3' end when viral RNA duplicates; Be that the template synthon is for positive chain RNA again with the minus strand.The nucleotide sequence of 3Dpol and aminoacid sequence have the conservative property of height.The present invention uses the RNAi technology and obtains obviously suppressing the shNRA sequence that FMDV duplicates in the cell levels screening.
Technical scheme of the present invention is achieved in that a kind of anti-pig O type hoof-and-mouth disease viral disease shRNA, it is characterized in that the method for synthetic foot-and-mouth disease virus resistant band shRNA carrier is following:
1. according to the O type foot and mouth disease genome sequence of announcing among the GenBank, select VP
1With the conservative section in the 3D gene, choose the long nucleotide sequence of one section 19bp; This sequence is 75bp after 5 ' and 3 ' non-translational region and initiator codon not, because modulin is rich in these zones, may influence the RNA interference effect;
2. to be undertaken by following requirement when choosing the 19bp nucleotide sequence:
1) calculate the GC content of this 19bp nucleotide sequence, GC content must be between 40%-60%.GC content is best about 45% o'clock greatly;
2) in the 15-19 site, have 3 A or T nucleic acid residue at least, can strengthen the RNA interferon activity;
3) guarantee that the 19bp nucleotide sequence can not form secondary structure;
4) between positive-sense strand and antisense strand oligonucleotide sequence, add intervening sequence TTCAAGAGA, make it can form hairpin structure, add the terminator sequence TTTTTT of RNA polymerase at the 3' of antisense strand end;
3.The 19bp nucleotide sequence is specific does not have too big similarity to Schweineseuche viral disease goal gene with other genes, according to carrier characteristics, and synthetic hairpin structure shRNA corresponding DNA sequences.
The sequence of described anti-pig O type hoof-and-mouth disease viral disease shRNA is following, and nucleotides sequence is wherein classified 5'-3' as;
pSIREN-FMDV-?VP
1
-1
Positive-sense strand:
GATCCAATTACTCAGACACTTGCAGTTTCAAGAGAACTGCAAGTGTCTGAGTAATTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAATTACTCAGACACTTGCAGTTCTCTTGAAACTGCAAGTGTCTGAGTAATTG
pSIREN-FMDV-?VP
1
-2
Positive-sense strand:
GATCCAAGTTAATGTGTTGGACCTGATTCAAGAGATCAGGTCCAACACATTAACTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGTTAATGTGTTGGACCTGATCTCTTGAATCAGGTCCAACACATTAACTTG
pSIREN-FMDV-?VP
1
-3
Positive-sense strand:
GATCCAACAGCTTACCACAAGGAACCTTCAAGAGAGGTTCCTTGTGGTAAGCTGTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAACAGCTTACCACAAGGAACCTCTCTTGAAGGTTCCTTGTGGTAAGCTGTTG
pSIREN-FMDV-?VP
1
-4
Positive-sense strand:
GATCCAAGGCAGAAAGAACTCTGCCTTTCAAGAGAAGGCAGAGTTCTTTCTGCCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGGCAGAAAGAACTCTGCCTTCTCTTGAAAGGCAGAGTTCTTTCTGCCTTG
pSIREN-FMDV-?VP
1
-5
Positive-sense strand:
GATCCAAGGCAACTCGTGTTACTGAATTCAAGAGATTCAGTAACACGAGTTGCCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGGCAACTCGTGTTACTGAATCTCTTGAATTCAGTAACACGAGTTGCCTTG
pSIREN-FMDV-?VP
1
-6
Positive-sense strand:
GATCCAAGAGAGCCGAGACATACTGTTTCAAGAGAACAGTATGTCTCGGCTCTCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGAGAGCCGAGACATACTGTTCTCTTGAAACAGTATGTCTCGGCTCTCTTG
pSIREN-FMDV-3D-1
Positive-sense strand:
GATCCATAGCGTCACCGAAGTTACTATTCAAGAGATAGTAACTTCGGTGACGCTATTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAATAGCGTCACCGAAGTTACTATCTCTTGAATAGTAACTTCGGTGACGCTATG
pSIREN-FMDV-3D-2
Positive-sense strand:
GATCCAAGACTCTAGTGAACACGGAGTTCAAGAGACTCCGTGTTCACTAGAGTCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGACTCTAGTGAACACGGAGTCTCTTGAACTCCGTGTTCACTAGAGTCTTG
pSIREN-FMDV-3D-3
Positive-sense strand:
GATCCAAGTGACTACGACCTGGACTTTTCAAGAGAAAGTCCAGGTCGTAGTCACTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGTGACTACGACCTGGACTTTCTCTTGAAAAGTCCAGGTCGTAGTCACTTG
pSIREN-FMDV-3D-4
Positive-sense strand:
GATCCAAGGCCTCTTCGAGATTCCAATTCAAGAGATTGGAATCTCGAAGAGGCCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGGCCTCTTCGAGATTCCAATCTCTTGAATTGGAATCTCGAAGAGGCCTTG
pSIREN-FMDV-3D-5
Positive-sense strand:
GATCCAAGCTACAGATCACTTTACCTTTCAAGAGAAGGTAAAGTGATCTGTAGCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGCTACAGATCACTTTACCTTCTCTTGAAAGGTAAAGTGATCTGTAGCTTG
pSIREN-FMDV-3D-6
Positive-sense strand:
GATCCAAGAGGACAAAGCGCTGTTTCTTCAAGAGAGAAACAGCGCTTTGTCCTCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGAGGACAAAGCGCTGTTTCTCTCTTGAAGAAACAGCGCTTTGTCCTCTTG
Above-mentioned shRNA is connected on the RNAi-Ready pSIREN-RetroQ-ZsGreen vector (available from clontech company); Then with in the big upgrading grain of interference carrier, linearizing, ethanol sedimentation, the transfection BHK-21 cell; Again cells transfected is infected O type foot and mouth disease virus; Receive poison then; And extraction venom RNA; Filter out the expression vector that efficient inhibition foot and mouth disease virus is duplicated through Real-time PCR at last, these carriers just have the sick ability of resisting O-type foot and mouth disease virus.
Described RNAi-Ready pSIREN-RetroQ-ZsGreen vector is as rna interference vector; At first the shRNA sequence of above-mentioned synthetic is closed chain; The condition of closing chain is 95 ℃ of 30s; 72 ℃ of 2min; 37 ℃ of 2min, 25 ℃ of 2min, these two strandss have sticky end; Can be directly connected on the interference carrier, the rna interference vector that the present invention screened contains the shRNA that fine inhibition foot and mouth disease virus is duplicated.
Positively effect of the present invention is through design shRNA; DNA with synthetic closes chain then; Be cloned on the RNAi-Ready pSIREN-RetroQ-ZsGreen vector; At last with transfection BHK-21 cell after the carrier linearizing; Utilize the shRNA that expresses to suppress duplicating of foot and mouth disease virus, thereby play the effect of prevention; In recent years; A lot of foot and mouth disease virus variants have constantly appearred again in China different areas; There are unsafe factors such as virulence reversion, inactivation of virus are not thorough, live virus escape vaccine source mill again because of foot and mouth disease virus; So prevent and control the popular more and more difficult that becomes of foot and mouth disease through immunization; And the present invention can disturb through RNA and prevents foot and mouth disease virus; This will exert far reaching influence to the development of pig industry, for the mankind bring bigger interests.
Description of drawings
Fig. 1 is vector rna i-Ready pSIREN-RetroQ-ZsGreen and is connected into the interference fragment structure iron.
Fig. 2 is that shRNA closes chain agarose gel electrophoresis figure as a result.
Fluorogram when Fig. 3 is expression plasmid transfection BHK-21 cell 22h.
Fig. 4 meets BHK-21 cytopathy figure after the malicious different time sections.
Fig. 5 is the extraction agarose gel electrophoresis figure as a result that attacks poison back cell total rna.
Fig. 6 is confidential reference items typical curve and sample standard curve plotting result.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.These embodiment are intended to further describe for example the present invention, rather than limit the present invention by any way.
(1), selects VP according to the O type foot and mouth disease genome sequence of announcing among the GenBank
1With the conservative section in the 3D gene, choose the long nucleotide sequence of one section 19bp.Design comprises the positive-sense strand and the antisense strand oligonucleotide sequence of goal gene siRNA sequence respectively, and the GC content of this nucleotide sequence is 40%-60%; In the 15-19 site, have 3 A or T nucleic acid residue at least, can strengthen the RNA interferon activity; Guarantee that the 19bp nucleotide sequence can not form secondary structure.
(2) between positive-sense strand and antisense strand oligonucleotide sequence, add intervening sequence TTCAAGAGA; Make it can form hairpin structure; Add the terminator sequence TTTTTT of RNA polymerase at the 3' of antisense strand end, after add ECORI and the adaptive sequence of BamHI restriction enzyme site; Entrust Shanghai to give birth to the synthetic that the biological company limited of worker carries out single stranded DNA, totally 120 pairs, comprising two out of order interference fragments.
(3) the 19bp nucleotide sequence is specific does not have too big similarity to Schweineseuche viral disease goal gene with other genes, according to carrier characteristics, and synthetic hairpin structure shRNA corresponding DNA sequences.
With the positive and negative adopted chain DNA fragment of synthetic, with deionized water dissolving, final concentration is 100 μ Mol/L, respectively gets behind the 10 μ l synthetic double-stranded on the PCR appearance: 95 ℃ of 30s, and 72 ℃ of 2min, 37 ℃ of 2min, 25 ℃ of 2min place the back on ice and store for future use for-20 ℃.Part is closed the chain result as shown in Figure 2.
pSIREN-FMDV-?VP
1
-1
5'-GATCCAAATTACTCAGACACTTGCAGTTTCAAGAGAACTGCAAGTGTCTGAGTAATTTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAAATTACTCAGACACTTGCAGTTCTCTTGAAACTGCAAGTGTCTGAGTAATTG-3'
pSIREN-FMDV-?VP
1
-2
5'-GATCCAAAGTTAATGTGTTGGACCTGATTCAAGAGATCAGGTCCAACACATTAACTTTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAAAGTTAATGTGTTGGACCTGATCTCTTGAATCAGGTCCAACACATTAACTTG-3'
pSIREN-FMDV-?VP
1
-3
5'-GATCCAAACAGCTTACCACAAGGAACCTTCAAGAGAGGTTCCTTGTGGTAAGCTGTTTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAAACAGCTTACCACAAGGAACCTCTCTTGAAGGTTCCTTGTGGTAAGCTGTTG-3'
pSIREN-FMDV-?VP
1
-4
5'-GATCCAAAGGCAGAAAGAACTCTGCCTTTCAAGAGAAGGCAGAGTTCTTTCTGCCTTTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAAAGGCAGAAAGAACTCTGCCTTCTCTTGAAAGGCAGAGTTCTTTCTGCCTTG-3'
pSIREN-FMDV-?VP
1
-5
5'-GATCCAAAGGCAACTCGTGTTACTGAATTCAAGAGATTCAGTAACACGAGTTGCCTTTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAAAGGCAACTCGTGTTACTGAATCTCTTGAATTCAGTAACACGAGTTGCCTTG-3'
pSIREN-FMDV-?VP
1
-6
5'-GATCCAAAGAGAGCCGAGACATACTGTTTCAAGAGAACAGTATGTCTCGGCTCTCTTTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAAAGAGAGCCGAGACATACTGTTCTCTTGAAACAGTATGTCTCGGCTCTCTTG-3'
pSIREN-FMDV-3D-1
5'-GATCCAATAGCGTCACCGAAGTTACTATTCAAGAGATAGTAACTTCGGTGACGCTATTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAATAGCGTCACCGAAGTTACTATCTCTTGAATAGTAACTTCGGTGACGCTATG-3'
pSIREN-FMDV-3D-2
5'-GATCCAAAGACTCTAGTGAACACGGAGTTCAAGAGACTCCGTGTTCACTAGAGTCTTTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAAAGACTCTAGTGAACACGGAGTCTCTTGAACTCCGTGTTCACTAGAGTCTTG-3'
pSIREN-FMDV-3D-3
5'-GATCCAAAGTGACTACGACCTGGACTTTTCAAGAGAAAGTCCAGGTCGTAGTCACTTTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAAAGTGACTACGACCTGGACTTTCTCTTGAAAAGTCCAGGTCGTAGTCACTTG-3'
pSIREN-FMDV-3D-4
5'-GATCCAAAGGCCTCTTCGAGATTCCAATTCAAGAGATTGGAATCTCGAAGAGGCCTTTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAAAGGCCTCTTCGAGATTCCAATCTCTTGAATTGGAATCTCGAAGAGGCCTTG-3'
pSIREN-FMDV-3D-5
5'-GATCCAAAGCTACAGATCACTTTACCTTTCAAGAGAAGGTAAAGTGATCTGTAGCTTTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAAAGCTACAGATCACTTTACCTTCTCTTGAAAGGTAAAGTGATCTGTAGCTTG-3'
pSIREN-FMDV-3D-6
5'-GATCCAAGAGGACAAAGCGCTGTTTCTTCAAGAGAGAAACAGCGCTTTGTCCTCTTTTTTTTACGCGTG-3'
5'-AATTCACGCGTAAAAAAAAGAGGACAAAGCGCTGTTTCTCTCTTGAAGAAACAGCGCTTTGTCCTCTTG-3'
The double-stranded shRNA that will anneal mixes with vector rna i-Ready pSIREN-RetroQ-ZsGreen and is connected.Connect product transformed competence colibacillus bacillus coli DH 5 alpha; The picking mono-clonal spends the night in the LB nutrient solution behind the coated plate; Extract bacterium liquid plasmid then, serve the sea and give birth to the order-checking of worker's biotechnology Services Co., Ltd, identify whether sequence successfully is connected to carrier and identifies whether shRNA is correct.
Behind little hamster kidney cell line (BHK-21) cell recovery, do not contain antibiotic DMEM nutritive medium and go down to posterity, place 37 ℃, 5%, CO with containing 10% calf serum
2Cultivate for use in the incubator.Growth and the titration of O type hoof-and-mouth disease poison strain OS-99 are all carried out through the BHK-21 cell.Titration is undertaken by following method; The BHK-21 cell is implanted 96 orifice plates the day before yesterday infecting; Former venom is done 10 times of dilutions of successive in the sterilization small test tube; Draw each dilution viral liquid 0.1ml; Be incorporated in the responsive BHK-21 cell that 96 holes have grown up to individual layer each 8 hole of dilution viral liquid inoculation.In 37 ℃ of static cultivations, observe day by day (generally needing about 7-10d), until terminal point, just see causing the high dilution of viral value-added virus.Survey proviral TCID with Reed-Muench formula and principle
50Should be 0.1ml10
-3.95The viral liquid of dilution.Look into antilogarithm, get 8913, promptly should equal 1 TCID by 8913 times of diluent 0.1ml of virus
50
Embodiment 6 anti-pig O type hoof-and-mouth disease viral disease shRNA transfection BHK-21 cells and attack poison experiment
With the expression vector enlarged culturing that builds, heavy dose of plasmid that extracts, ethanol sedimentation after the linearizing is established transfection expression plasmid group, negative control group, non-transfection group and blank normal cell group as shown in Figure 3 then on 24 orifice plates.Do not contain antibiotic DMEM nutritive medium and go down to posterity the BHK-21 cell in 24 well culture plates, every hole 5.0 * 10 with containing 10% calf serum
4Cell/500 μ l, 37 ℃, 5%, CO
2Cultivate 20-24 h in the incubator, make cell attachment growth full scale reach 90-95%, according to Transfectamine2000
TMReagent reagent specification sheets operation steps is carried out transfection.Behind the BHK-21 cell cultures 20-24 h of transfection expression plasmid, with 2 * 10
3TCID
50The FMDV in/hole infects, continue to cultivate, respectively at connect observe CPE behind poison 10,20,40,80 h variation as shown in Figure 4 and the cell venom-80 of collecting different time sections ℃ store for future use.
Embodiment 7 attacks extraction and the Real-time PCR of poison cell RNA
Attacked malicious BHK-21 cell from-80 ℃ of taking-ups, extracted cell RNA as shown in Figure 5 with TRIZOL reagent.Get the RNA that 5 μ l DNase I are handled, carry out conventional reverse transcription reaction with test kit and make the RNA reverse transcription become cDNA.Design VP
1, 3D, β-actin gene order (table 1), to clone correct VP
1, 3D and β-actin plasmid be template, respectively sets up 6 10 times of gradient dilutions, render target gene and internal control gene typical curve are as shown in Figure 6.Display-object gene and internal control gene relation conefficient are 0.998 as a result, 6 points are described all point-blank, and linear relationship is good, and accuracy is than higher.
The active detected result of embodiment 8 anti-pig O type hoof-and-mouth disease viral disease shRNA
Target gene and internal control gene sample are respectively done three repetitions; Get the mean number of Ct value; The result who the mean value of sample Ct value is brought into the sample standard curve brings the result of β-actin typical curve into divided by the β-mean number of actin Ct value; Draw the expression amount of sample; The expression amount of the big more virus of value is just few more; Illustrate that the shRNA anti-virus ability is strong; Compare with negative control through calculation result; Interference plasmid group genetic expression inhibiting rate between 63% to 96.8%, pSIREN-FMDV-Green-VP wherein
1-4 suppress efficient with two expression plasmids of pSIREN-FMDV-Green-3D-2 preferably is respectively 96.8% and 87%.
VP1-1 positive-sense strand: 5'-gatccAATTACTCAGACACTTGCAGTTTCAAGAGAACTGCAAGTGTCTGAGTA ATTTTTTTTACGCGTg-----3'
VP1-1 antisense strand: 5'-aattcACGCGTAAAAAAAATTACTCAGACACTTGCAGTTCTCTTGAAACTGCA AGTGTCTGAGTAATTg-----3'
VP1-2 positive-sense strand: 5'-gatccAAGTTAATGTGTTGGACCTGATTCAAGAGATCAGGTCCAACACATTAA CTTTTTTTTACGCGTg-----3'
VP1-2 antisense strand: 5'-aattcACGCGTAAAAAAAAGTTAATGTGTTGGACCTGATCTCTTGAATCAGGT CCAACACATTAACTTg-----3'
VP1-3 positive-sense strand: 5'-gatccAACAGCTTACCACAAGGAACCTTCAAGAGAGGTTCCTTGTGGTAAGCT GTTTTTTTTACGCGTg-----3'
VP1-3 antisense strand: 5'-aattcACGCGTAAAAAAAACAGCTTACCACAAGGAACCTCTCTTGAAGGTTCC TTGTGGTAAGCTGTTg-----3'
VP1-4 positive-sense strand: 5'-gatccAAGGCAGAAAGAACTCTGCCTTTCAAGAGAAGGCAGAGTTCTTTCTGC CTTTTTTTTACGCGTg-----3'
VP1-4 antisense strand: 5'-aattcACGCGTAAAAAAAAGGCAGAAAGAACTCTGCCTTCTCTTGAAAGGCAG AGTTCTTTCTGCCTTg-----3'
VP1-5 positive-sense strand: 5'-gatccAAGGCAACTCGTGTTACTGAATTCAAGAGATTCAGTAACACGAGTTGC CTTTTTTTTACGCGTg-----3'
VP1-5 antisense strand: 5'-aattcACGCGTAAAAAAAAGGCAACTCGTGTTACTGAATCTCTTGAATTCAGT AACACGAGTTGCCTTg-----3'
VP1-6 positive-sense strand: 5'-gatccAAGAGAGCCGAGACATACTGTTTCAAGAGAACAGTATGTCTCGGCTCT CTTTTTTTTACGCGTg-----3'
VP1-6 antisense strand: 5'-aattcACGCGTAAAAAAAAGAGAGCCGAGACATACTGTTCTCTTGAAACAGTA TGTCTCGGCTCTCTTg-----3'
3D-1 positive-sense strand: 5'-gatccATAGCGTCACCGAAGTTACTATTCAAGAGATAGTAACTTCGGTGACGC TATTTTTTTACGCGTg-----3'
3D-1 antisense strand: 5'-aattcACGCGTAAAAAAATAGCGTCACCGAAGTTACTATCTCTTGAATAGTAA CTTCGGTGACGCTATg-----3'
3D-2 positive-sense strand: 5'-gatccAAGACTCTAGTGAACACGGAGTTCAAGAGACTCCGTGTTCACTAGAGT CTTTTTTTTACGCGTg-----3'
3D-2 antisense strand: 5'-aattcACGCGTAAAAAAAAGACTCTAGTGAACACGGAGTCTCTTGAACTCCGT GTTCACTAGAGTCTTg-----3'
3D-3 positive-sense strand: 5'-gatccAAGTGACTACGACCTGGACTTTTCAAGAGAAAGTCCAGGTCGTAGTCA CTTTTTTTTACGCGTg-----3'
3D-3 antisense strand: 5'-aattcACGCGTAAAAAAAAGTGACTACGACCTGGACTTTCTCTTGAAAAGTCC AGGTCGTAGTCACTTg-----3'
3D-4 positive-sense strand: 5'-gatccAAGGCCTCTTCGAGATTCCAATTCAAGAGATTGGAATCTCGAAGAGGC CTTTTTTTTACGCGTg-----3'
3D-4 antisense strand: 5'-aattcACGCGTAAAAAAAAGGCCTCTTCGAGATTCCAATCTCTTGAATTGGAA TCTCGAAGAGGCCTTg-----3'
3D-5 positive-sense strand: 5'-gatccAAGCTACAGATCACTTTACCTTTCAAGAGAAGGTAAAGTGATCTGTAG CTTTTTTTTACGCGTg-----3'
3D-5 antisense strand: 5'-aattcACGCGTAAAAAAAAGCTACAGATCACTTTACCTTCTCTTGAAAGGTAA AGTGATCTGTAGCTTg-----3'
3D-6 positive-sense strand: 5'-gatccAAGAGGACAAAGCGCTGTTTCTTCAAGAGAGAAACAGCGCTTTGTCCT CTTTTTTTTACGCGTg-----3'
3D-6 antisense strand: 5'-aattcACGCGTAAAAAAAAGAGGACAAAGCGCTGTTTCTCTCTTGAAGAAACA GCGCTTTGTCCTCTTg-----3'
Claims (4)
1. anti-pig O type hoof-and-mouth disease viral disease shRNA is characterized in that the method for synthetic foot-and-mouth disease virus resistant band shRNA carrier is following:
According to the O type foot and mouth disease genome sequence of announcing among the GenBank, select VP
1With the conservative section in the 3D gene, choose the long nucleotide sequence of one section 19bp; This sequence is 75bp after 5 ' and 3 ' non-translational region and initiator codon not, because modulin is rich in these zones, may influence the RNA interference effect;
To be undertaken by following requirement when choosing the 19bp nucleotide sequence:
1) calculate the GC content of this 19bp nucleotide sequence, GC content must be between 40%-60%, and GC content is best about 45% o'clock greatly;
2) in the 15-19 site, have 3 A or T nucleic acid residue at least, can strengthen the RNA interferon activity;
3) guarantee that the 19bp nucleotide sequence can not form secondary structure;
4) between positive-sense strand and antisense strand oligonucleotide sequence, add intervening sequence TTCAAGAGA, make it can form hairpin structure, add the terminator sequence TTTTTT of RNA polymerase at the 3' of antisense strand end;
The 19bp nucleotide sequence is specific does not have too big similarity to Schweineseuche viral disease goal gene with other genes, according to carrier characteristics, and synthetic hairpin structure shRNA corresponding DNA sequences.
2. a kind of anti-pig O type hoof-and-mouth disease viral disease shRNA according to claim 1 is characterized in that the sequence of described anti-pig O type hoof-and-mouth disease viral disease shRNA is following, and nucleotides sequence is wherein classified 5'-3' as;
pSIREN-FMDV-VP1-1
Positive-sense strand:
GATCCAATTACTCAGACACTTGCAGTTTCAAGAGAACTGCAAGTGTCTGAGTAATTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAATTACTCAGACACTTGCAGTTCTCTTGAAACTGCAAGTGTCTGAGTAATTG
pSIREN-FMDV-VP1-2
Positive-sense strand:
GATCCAAGTTAATGTGTTGGACCTGATTCAAGAGATCAGGTCCAACACATTAACTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGTTAATGTGTTGGACCTGATCTCTTGAATCAGGTCCAACACATTAACTTG
pSIREN-FMDV-VP1-3
Positive-sense strand:
GATCCAACAGCTTACCACAAGGAACCTTCAAGAGAGGTTCCTTGTGGTAAGCTGTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAACAGCTTACCACAAGGAACCTCTCTTGAAGGTTCCTTGTGGTAAGCTGTTG
pSIREN-FMDV-VP1-4
Positive-sense strand:
GATCCAAGGCAGAAAGAACTCTGCCTTTCAAGAGAAGGCAGAGTTCTTTCTGCCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGGCAGAAAGAACTCTGCCTTCTCTTGAAAGGCAGAGTTCTTTCTGCCTTG
pSIREN-FMDV-VP1-5
Positive-sense strand:
GATCCAAGGCAACTCGTGTTACTGAATTCAAGAGATTCAGTAACACGAGTTGCCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGGCAACTCGTGTTACTGAATCTCTTGAATTCAGTAACACGAGTTGCCTTG
pSIREN-FMDV-VP1-6
Positive-sense strand:
GATCCAAGAGAGCCGAGACATACTGTTTCAAGAGAACAGTATGTCTCGGCTCTCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGAGAGCCGAGACATACTGTTCTCTTGAAACAGTATGTCTCGGCTCTCTTG
pSIREN-FMDV-3D-1
Positive-sense strand:
GATCCATAGCGTCACCGAAGTTACTATTCAAGAGATAGTAACTTCGGTGACGCTATTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAATAGCGTCACCGAAGTTACTATCTCTTGAATAGTAACTTCGGTGACGCTATG
pSIREN-FMDV-3D-2
Positive-sense strand:
GATCCAAGACTCTAGTGAACACGGAGTTCAAGAGACTCCGTGTTCACTAGAGTCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGACTCTAGTGAACACGGAGTCTCTTGAACTCCGTGTTCACTAGAGTCTTG
pSIREN-FMDV-3D-3
Positive-sense strand:
GATCCAAGTGACTACGACCTGGACTTTTCAAGAGAAAGTCCAGGTCGTAGTCACTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGTGACTACGACCTGGACTTTCTCTTGAAAAGTCCAGGTCGTAGTCACTTG
pSIREN-FMDV-3D-4
Positive-sense strand:
GATCCAAGGCCTCTTCGAGATTCCAATTCAAGAGATTGGAATCTCGAAGAGGCCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGGCCTCTTCGAGATTCCAATCTCTTGAATTGGAATCTCGAAGAGGCCTTG
pSIREN-FMDV-3D-5
Positive-sense strand:
GATCCAAGCTACAGATCACTTTACCTTTCAAGAGAAGGTAAAGTGATCTGTAGCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGCTACAGATCACTTTACCTTCTCTTGAAAGGTAAAGTGATCTGTAGCTTG
pSIREN-FMDV-3D-6
Positive-sense strand:
GATCCAAGAGGACAAAGCGCTGTTTCTTCAAGAGAGAAACAGCGCTTTGTCCTCTTTTTTTTACGCGTG
Antisense strand:
AATTCACGCGTAAAAAAAAGAGGACAAAGCGCTGTTTCTCTCTTGAAGAAACAGCGCTTTGTCCTCTTG。
3. a kind of anti-pig O type hoof-and-mouth disease viral disease shRNA according to claim 1; It is characterized in that described shRNA carrier construction method is for to be connected to above-mentioned shRNA on the RNAi-Ready pSIREN-RetroQ-ZsGreen vector; Then with the big upgrading grain of interference carrier; Linearizing; Ethanol sedimentation; In the transfection BHK-21 cell; Again cells transfected is infected O type foot and mouth disease virus; Receive poison then; And extraction venom RNA; Filter out the expression vector that efficient inhibition foot and mouth disease virus is duplicated through Real-time PCR at last, these carriers just have the sick ability of resisting O-type foot and mouth disease virus.
4. a kind of anti-pig O type hoof-and-mouth disease viral disease shRNA according to claim 3; It is characterized in that described RNAi-Ready pSIREN-RetroQ-ZsGreen vector is as rna interference vector; At first the shRNA sequence of above-mentioned synthetic is closed chain; The condition of closing chain is 95 ℃ of 30s; 72 ℃ of 2min; 37 ℃ of 2min; 25 ℃ of 2min; These two strandss have sticky end; Can be directly connected on the interference carrier, the rna interference vector that the present invention screened contains the shRNA that fine inhibition foot and mouth disease virus is duplicated.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703392A (en) * | 2012-06-12 | 2012-10-03 | 石河子大学 | Preparation method of transgenically cloned pig integrating O-type foot and mouth disease virus shRNA (Short Hairpin Ribonucleic Acid) |
| CN102703504A (en) * | 2012-07-02 | 2012-10-03 | 复旦大学 | shRNA (short hairpin ribonucleic acid) transgene recombinant plasmid capable of inhibiting foot and mouth disease virus and application thereof |
| CN108456692A (en) * | 2018-01-30 | 2018-08-28 | 广东省农业科学院动物卫生研究所 | A kind of the tetrad miRNA and construction method of foot-and-mouth disease virus resistant infection |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002499A (en) * | 2010-10-14 | 2011-04-06 | 南京农业大学 | shRNA capable of inhibiting replication and infection of Japanese encephalitis virus and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102002499A (en) * | 2010-10-14 | 2011-04-06 | 南京农业大学 | shRNA capable of inhibiting replication and infection of Japanese encephalitis virus and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《中国兽医学报》 20100531 崔丽瑾等 靶向IRES 序列的siRNA 对口蹄疫病毒体外增殖抑制效果 第629-633页 1-4 第30卷, 第5期 * |
| 《中国预防兽医学报》 20060131 娄高明等 6株猪O型口蹄疫病毒VP1基因的克隆与序列分析 第72-76页 1-4 第28卷, 第1期 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703392A (en) * | 2012-06-12 | 2012-10-03 | 石河子大学 | Preparation method of transgenically cloned pig integrating O-type foot and mouth disease virus shRNA (Short Hairpin Ribonucleic Acid) |
| CN102703504A (en) * | 2012-07-02 | 2012-10-03 | 复旦大学 | shRNA (short hairpin ribonucleic acid) transgene recombinant plasmid capable of inhibiting foot and mouth disease virus and application thereof |
| CN102703504B (en) * | 2012-07-02 | 2015-04-22 | 复旦大学 | shRNA (short hairpin ribonucleic acid) transgene recombinant plasmid capable of inhibiting foot and mouth disease virus and application thereof |
| CN108456692A (en) * | 2018-01-30 | 2018-08-28 | 广东省农业科学院动物卫生研究所 | A kind of the tetrad miRNA and construction method of foot-and-mouth disease virus resistant infection |
| CN108456692B (en) * | 2018-01-30 | 2022-06-14 | 广东省农业科学院动物卫生研究所 | Quadruple miRNA for resisting foot-and-mouth disease virus infection and construction method |
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