CN102002499B - shRNA capable of inhibiting replication and infection of Japanese encephalitis virus and application thereof - Google Patents
shRNA capable of inhibiting replication and infection of Japanese encephalitis virus and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering and discloses shRNA capable of inhibiting replication and infection of Japanese encephalitis virus and application thereof. The shRNA takes a highly-conserved sequence in a Japanese encephalitis virus genome as a target sequence which is SEQIDNO.9, SEQIDNO.10, SEQIDNO.11 or SEQIDNO.12. The shRNA eukaryotic expression plasmid can be obtained by connecting a template of the shRNA with a linearized vector pGPU6/GFP/Neo and respectively aims at Japanese encephalitis virus E and NS3 or NS4b gene mRNA sequence. The shRNA and the shRNA eukaryotic expression plasmid provided by the invention can be applied to a medicament for treating and/or preventing JEV (Japanese Encephalitis Virus) infection and provides a novel selectable medicament for treating and preventing the JEV infection.
Description
Technical field
The invention belongs to gene engineering technology field, relate to and a kind ofly can suppress shRNA and the application thereof that encephalitis b virus duplicates and infects.
Background technology
(Japanese encephalitis is by encephalitis b virus (Japanese encephalitis virus, a kind of serious infecting both domestic animals and human arthropod borne viral disease that JEV) causes JE) to encephalitis B.This disease endangers huge to the mankind, be one of modal vector borne diseases of human cns, is distributed widely in the Asia, particularly some countries and regions, the Far East.Its popular distribution range constantly enlarges in recent years, and popular frequency also constantly strengthens.
Encephalitis b virus belongs to the flaviviridae Flavivirus.Virus particle is 20 body symmetries, is surrounded by fine and close lipoprotein cyst membrane in the nucleic acid outside.The JEV genome is a sub-thread positive chain RNA molecule, and itself has infectivity.Its genome encoding product is 3 structural protein (C, prM, E) and 7 Nonstructural Proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).
Encephalitis belongs to disease of natural focus, and mosquito is the main communication media of this disease.Generation and climatope that this is sick have confidential relation, are obvious seasonal.The general epidemic peak of China was at 7,8,9 three months.Multiple animal all can infect this disease, becomes contagium after the infection.Find that on inspection in popular lesion, the inapparent infection rate of livestock and poultry is all very high, particularly secondly pig is Ma Heniu.Virus RT in infection animal blood is very short, mainly be present in the testis of cns and swelling, but pig infects the back viremia phase length of holding time, and virus titer is high in the blood, and the propagation of encephalitis is played an important role.Simultaneously, pig is that encephalitis is propagated to human most important host, and the infection of JEV is pig-mosquito-people's chain generally speaking.The morning and evening of pig infection encephalitis time and the height of infection rate are popular closely related with people's encephalitis.After the infection, tangible encephalitis clinical symptom appears in people, monkey, horse and donkey, and case fatality rate is higher.The encephalitis symptom does not take place in swinery, and farrowing sow can show high heat, miscarriage, stillborn foetus and mummy tire, and the brain defective symptom appears in the stillbirth fetus, and testitis then appears in boar, and development brings tremendous loss to pig industry.
RNA disturbs that (RNA interference RNAi) is meant the sequence-specific gene silencing phenomenon of double chain RNA mediate.The RNA interference comes to light in plant and unicellular lower eukaryote at first, along with going deep into of research, recently in high eukaryote, also comes to light, and proves a kind of important evolution conservative phenomenon.Proved that short bifilar RNA interfering (siRNA) is by the special endonuclease of dsRNA, the Dicer enzyme is sheared.It is to be combined to constitute by siRNA and a kind of multienzyme complex that RNA induces reticent mixture (RISC), and it is positioned at the specific position of mRNA, and the activity effect mRNA of performance endonuclease and excision enzyme.Except the gene silencing (degraded mRNA) at transcriptional level, siRNA also is proved to be through the reticent promotor of dna methylation and reduces protein expression.Short hairpin RNA (shRNA) utilizes the carrier transfered cell usually, and can be passed in the daughter cell and go, thereby makes the gene silencing can be by heredity.The hairpin structure of shRNA can be cut into siRNA by cell mechanism, reaches the purpose of degraded mRNA then by above-mentioned mechanism.The validity that its biggest advantage is to have height is with specificity and have and defend fast and result of treatment.Its treatment field that acts on gene functional research field and the various diseases especially treatment field of virus disease has demonstrated immeasurable value; For example at anti AIDS virus (HIV); Hepatitis C virus (HCV); Find all in the research of hepatitis B virus (HBV) and poliovirus viruses such as (Poliovirus) that RNAi is fabulous for the print effect that suppresses this viroid, maybe be new for the treatment foundation of this viroid disease, more effective approach.Undoubtedly, along with to the understanding in depth and technology further perfect of RNAi mechanism, the RNAi technology will be brought new revolution for the control of life science and clinical disease.
The popular of encephalitis B is one of global public health problem.Still do not have the specific treatment means for encephalitis at present, therefore prevent the generation of encephalitis particularly important.And traditional vaccine production can not be satisfied human growing needs, thereby the scientific research personnel constantly seeks new therapeutic modality.This emerging technology of RNAi promptly is wherein quite potential new way.At present; Utilization RNAi treatment encephalitis also is in the theoretical investigation of scientific research institutions both at home and abroad; Do not put in the clinical treatment on a large scale, but its result of study demonstrates the remarkable inhibition virus replication and the ability of infection, and great potentiality and good prospect are arranged in clinical treatment.
Summary of the invention
It is quick, special and significantly suppress shRNA and the application thereof that JEV duplicates and infects in zooblast and individuality to the purpose of this invention is to provide a kind of ability.
Another object of the present invention provides eukaryon expression plasmid and the application thereof that contains this shRNA.
The object of the invention can be realized through following technical scheme:
A kind ofly can suppress the shRNA that encephalitis b virus duplicates and infects; As its target sequence, described target sequence is: SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 with conservative E, NS3 and the NS4b gene order of JEV genome camber.
The template sequence of shRNA according to the invention is following:
ShRNA-E1: positive-sense strand template sequence SEQ ID NO.1, antisense strand template sequence SEQ ID NO.2;
ShRNA-E2: positive-sense strand template sequence SEQ ID NO.3, antisense strand template sequence SEQ ID NO.4;
ShRNA-NS3: positive-sense strand template sequence SEQ ID NO.5, antisense strand template sequence SEQ ID NO.6;
ShRNA-NS4b: positive-sense strand template sequence SEQ ID NO.7, antisense strand template sequence SEQ ID NO.8.
Wherein the target-gene sequence that is directed against of shRNA-E1 is SEQ ID NO.9; The target-gene sequence that shRNA-E2 is directed against is SEQ ID NO.10; The target-gene sequence that shRNA-NS3 is directed against is SEQ ID NO.11, and the target-gene sequence that shRNA-NS4b is directed against is SEQ ID NO.12.
A kind of shRNA eukaryon expression plasmid of target encephalitis b virus genome different zones promptly is that the template with shRNA of the present invention is connected with linearizing carrier pGPU6/GFP/Neo and obtains respectively being abbreviated as pGP-E1, pGP-E2, pGP-NS3 and pGP-NS4b respectively to the shRNA eukaryon expression plasmid of encephalitis b virus E, NS3 or NS4b gene mRNA sequence.
Inhibition encephalitis b virus of the present invention duplicate with the shRNA that infects preparation suppress that encephalitis b virus duplicates and/or the medicine that infects in application.
The shRNA eukaryon expression plasmid of target encephalitis b virus genome different zones of the present invention preparation suppress that encephalitis b virus duplicates and/or the medicine that infects in application.
Beneficial effect of the present invention:
Elder generation's transfection shRNA eukaryon expression plasmid of the present invention in zooblast; Carry out a series of tests (Fig. 4 ~ Fig. 8) with the virus attack cell again; The result shows that shRNA of the present invention can suppress JEV duplicating on cell effectively, and pair cell has good protective action.
Earlier shRNA eukaryon expression plasmid of the present invention is injected in the animal body, use the virus attack animal body (Figure 10 A, B) of various dose again; Perhaps the virus of shRNA eukaryon expression plasmid of the present invention and various dose is injected (Figure 10 C, D) in the animal body simultaneously; The result shows that shRNA of the present invention all can reduce the M & M of animal effectively; The animal dis motility rate reaches more than 50%, is significantly higher than noiseless effect group.Thus it is clear that, the effect that the shRNA eukaryon expression plasmid of the present invention effect that existing good preventing JEV infects on the animal individual level also has treatment JEV to infect.
In sum, shRNA provided by the present invention and shRNA eukaryon expression plasmid can treat and/or prevent in the medicine that JEV infects in preparation and use, and for treatment and prevention JEV infect a kind of new optional medicine are provided.
Description of drawings
Selected conservative section synoptic diagram among Fig. 1 JEV genome structure and the present invention.The arrow place is the selected target gene of shRNA site.
Fig. 2 pGPU6/GFP/Neo carrier structure figure.
The enzyme of Fig. 3 shRNA eukaryon expression plasmid is cut evaluation.M:lamda/
Eco130I; A1-5 and B1-5 are respectively pGP-E1 and pGP-E2
BbsThe I enzyme is cut the result; A ' 1-5 and B ' 1-5 are respectively pGP-E1 and pGP-E2
BamThe HI enzyme is cut the result; C1-5 and D1-5 are respectively pGP-NS3 and pGP-NS4b
BbsThe I enzyme is cut the result; C ' 1-5 and D ' 1-5 are respectively pGP-NS3 and pGP-NS4b
BamThe HI enzyme is cut the result.
Fig. 4 indirect immunofluorescence result.A, B, C, D, E, F are respectively pGP-E1, pGP-E2, pGP-NS3, pGP-NS4b, pGP-NC and Mock group result.
The TCID of the cell culture fluid of Fig. 5 after the shRNA effect
50Mensuration result.
Fig. 6 flow cytometry result.A, B, C, D, E, F are respectively pGP-E1, pGP-E2, pGP-NS3, pGP-NS4b, pGP-NC and Mock group result.
Fig. 7 Real time RT-PCR result.The relative expression quantity of mRNA is meant through the expression amount of 3 ' section non-coding region of goal gene of JEV and the ratio of negative control group destination gene expression amount after the shRNA interference effect.
Fig. 8 Western blotting result.Swimming lane 1 ~ 6 is respectively pGP-E1, pGP-E2, pGP-NS3, pGP-NS4b, pGP-NC and Mock group result.
Fig. 9 immunohistochemical methods result.A, B, C, D, E, F are respectively pGP-E1, pGP-E2, pGP-NS3, pGP-NS4b, Virus control and Mock group result.
Figure 10 protection of animal property experimental result.A, B are respectively earlier and attack malicious 20LD behind the injection shRNA eukaryon expression plasmid
50And 100LD
50JEV (NJ2008) group; C, D are respectively and inject shRNA eukaryon expression plasmid and 20LD simultaneously
50JEV (NJ2008) group is injected shRNA eukaryon expression plasmid and 100LD simultaneously
50JEV (NJ2008) group.
Embodiment
The preparation of embodiment 1 shRNA
(1) design of oligonucleotide is with synthetic
E, NS3, NS4b gene mRNA sequence with JEV are target sequence (Fig. 1); Utilize the siRNA design software of Ambion company; Through the homology analysis, synthesize 4 pairs of shRNA oligonucleotide chains to 1553 ~ 1571bp (E1), 1679 ~ 1697bp (E2), 6083 ~ 6101bp (NS3) and 7350 ~ 7368bp (NS4b) design of JEV gene respectively.Its loop structure has selected for use TTCAAGAGA to avoid forming termination signal, and the transcription termination sequence of shRNA adopts the T6 structure.5 ' end of positive-sense strand template has added CACC, cuts the cohesive end complementation that the back forms with the BbsI enzyme; 5 ' end of antisense strand template has added GATC, cuts the cohesive end complementation that the back forms with the BamHI enzyme; If first base of the target sequence of shRNA is not G, then behind the CACC of its template strand, add a G.The template strand sequence of four shRNA that designed is following: shRNA-E1: positive-sense strand template SEQ ID NO. 1, antisense strand template SEQ ID NO. 2; ShRNA-E2: positive-sense strand template SEQ ID NO.3, antisense strand template SEQ ID NO. 4; ShRNA-NS3: positive-sense strand template SEQ ID NO. 5, antisense strand template SEQ ID NO. 6; ShRNA-NS4b: positive-sense strand template SEQ ID NO. 7, antisense strand template SEQ ID NO. 8.Above-mentioned sequence is synthetic entrusts Shanghai JiMa pharmacy Technology Co., Ltd to carry out.
(2) annealing of shRNA template
ShRNA template strand is used TE (pH8.0) dissolving respectively, and concentration is 100 μ M.Get corresponding positive-sense strand template and antisense strand template solution, according to table 1 proportioning configuration annealing reaction system.
Table 1
| Component | Volume (μ l) |
| The |
5 |
| Positive-sense strand template (100 μ M) | 5 |
| Antisense strand template (100 μ M) | 5 |
| ddH 2O | 35 |
| Add up to | 50 |
On the PCR appearance, carry out anneal: 95 C 5min according to following program; 85 C 5min; 75 C 5min; 70 C 5min; 4 C preserve.Obtaining concentration after the anneal is the shRNA template of 10 μ M.With 500 times of gained template solution dilutions, final concentration is 20 nM, is used for ligation.
(3) linearizing of pGPU6/GFP/Neo carrier
Get 2 μ gpGPU6/GFP/Neo carriers (, seeing Fig. 2), carry out enzyme according to table 2 system and cut processing available from Shanghai Ji Ma company:
Table 2
| Component | Volume (μ l) |
| 10× |
5 |
| |
2 |
| |
2 |
| pGPU6/GFP/ |
2 μg |
| ddH 2O | To 50 μ l |
| Add up to | 50 |
37 C enzymes were cut 1 hour, and agarose electrophoresis uses Agarose Gel DNA Purification Kit Ver2.0 (TaKaRa) to reclaim electrophoresis detection estimated concentration, weaker concn to 50 ng/ μ l.
(4) structure of pGPHI/GFP/Neo-shRNA carrier
With the shRNA template of the present invention after the renaturation respectively with
BbsI with
BamThe linearizing pGPU6/GFP/Neo carrier of HI double digestion connects; Transformed into escherichia coli DH5 α (available from TAKARA company); Through plasmid extract, enzyme is cut and the evaluation and screening that checks order goes out positive plasmid, abbreviates pGP-E1 (corresponding shRNA-E1), pGP-E2 (corresponding shRNA-E2), pGP-NS3 (corresponding shRNA-NS3) and pGP-NS4b (corresponding shRNA-NS4b) respectively as.Choose the shNC (available from Shanghai Ji Ma company) that does not have any homologous sequence with JEV and musculus cdna group, its positive-sense strand template sequence is SEQ ID NO. 13, and its antisense strand template sequence is SEQ ID NO. 14; Be connected with the pGPU6/GFP/Neo carrier, abbreviate pGP-NC as in this shNC expression plasmid literary composition, be made as negative control.
A. carry out the ligation of carrier according to table 3 system:
Table 3
| Component | Volume (μ l) |
| 10 * T4 connects damping |
2 |
| pGPU6/GFP/Neo( Bbs I+ BamH I) | 1 |
| ShRNA template (20nM) | 1 |
| T4 dna ligase (5 weissU/ μ l) | 1 |
| ddH 2O | 15 |
| Add up to | 20 |
22 C 1hr, transformed into escherichia coli DH5 α..
B. 5 bacterium colonies of each ligation picking are inoculated into the LB that contains 50 μ g/ml kantlex and cultivate concentrated.
C. use alkaline lysis extracting plasmid, the gained plasmid is used
BamH I, Pst I enzyme respectively cut evaluation (Fig. 3).Positive recombinant vectors should by
BamH I cuts, and can not be cut by Pst I.
2.shRNA on cell levels, suppress duplicating of JEV
Get same amount (96 orifice plates, 0.2 μ g/ hole; 24 orifice plates, 0.8 μ g/ hole; 6 orifice plates, 4.0 μ g/ holes) pGP-E1 of the present invention, pGP-E2, pGP-NS3, pGP-NS4b and pGP-NC expression plasmid; With liposome method (Lipfectin2000, Invitrogen Group) difference transfection BHK21 cell (available from Shanghai cell institute), inoculate 100LD behind the 24h more respectively
50JEV (NJ2008, GeneBank:GQ918133).Carry out indirect immunofluorescence assay, TCID behind the virus infection 48h respectively
50Mensuration, flow cytometry, Real time RT-PCR, Western Blotting.More than organize negative contrast with the pGP-NC effect in the test, noiseless effect is not infected the BHK21 groups of cells of JEV as blank (Mock group).
(1) indirect immunofluorescence: cell to be detected with PBS washing 3 times, is waited to do the back with the fixing 1hr of 4 ℃ of absolute ethyl alcohols, resist (mouse-anti JEV E monoclonal antibody with successively then; Ministry of Agriculture's animal epidemic diagnosis provides with immune emphasis open laboratory, down together) and two anti-(sheep anti-mouse igg-FITC, doctor's moral biotech firms; Down together) 37 ℃ respectively act on 1hr; During this time with PBS washing 3 times, with after the two anti-reactions again with PBS washing 3 times, directly microscopy (Fig. 4) under fluorescent microscope.
The result shows; The fluorocyte digital display work of shRNA expression plasmid effect group of the present invention is less than negative control group (pGP-NC group); Fluorescence intensity also is weaker than negative control, and wherein pGP-E1, pGP-E2 group naked eyes almost can't see fluorescence, and visible its has very strong inhibition JEV replication.
(2) virus titer is measured: after will cultivating viral cell freeze thawing 3 times, get 100 μ l viral supernatant liquid and join 900 μ l and keep in the nutrient solution, get 100 μ l behind the mixing and be added to next and manage in the 900 μ l nutritive mediums, method is carried out 10 times of doubling dilutions to viral liquid according to this; The 96 porocyte plates of planting the BHK21 cell previous day are carried in taking-up, discard nutritive medium in the plate, play every hole 100 μ l, 8 repetitions of each extent of dilution by high dilution then; 37 ℃ of 5%CO
2Cultivate, observe the CPE production every day, calculate the TCID of virus according to the Reed-Muench method
50(Fig. 5).
As shown in the figure, the viral TCID after the effect of shRNA expression plasmid
50Descend 10 in negative control group (pGP-NC group)
3~ 10
4Doubly.
(3) flow cytometry: trypsin digestion cell becomes single, pours the EP pipe into, and PBS washes 2 times, and is centrifugal; The 3%BSA re-suspended cell, 4 ℃ of lucifuge sealing 1hr are centrifugal; 4 ℃ of lucifuges of 2% Superlysoform are 1hr fixedly, and are centrifugal; The 0.2%Tween-20 re-suspended cell, 37 ℃ of water-bath 15min are centrifugal; Resist with two of one anti-(mouse-anti JEV E monoclonal antibody) and FITC mark successively then that (sheep anti-mouse igg-FITC) respectively acts on 0.5 hr for 37 ℃; During this time with PBS washing 3 times; With after the two anti-reactions again with PBS washing 3 times, centrifugal, resuspended with 500 μ lPBS; Lucifuge detects (Fig. 6) with flow cytometer.
As shown in the figure, the ratio that the photogenic cell number accounts for total cell count in pGP-E1, pGP-E2, pGP-NS3 and the pGP-NS4b group is respectively 8.3%, 11.3%, 26.5%, 18.3% and significantly is lower than 99% of negative control group.
（4）Real?time?RT-PCR：
Cell culture in a, the taking-up 24 porocyte plates, (Qiagen) carries RNA with the total RNA extraction reagent box;
B, to advance cDNA according to table 4 system synthetic:
Table 4
| Component | Volume (μ l) |
| 5×PrimeScript TMDamping fluid | 2 |
| PrimeScript TMRT Enzyme Mix | 0.5 |
| Auele Specific Primer SEQ ID NO. 16 | 0.2 |
| |
3 |
| RNase Free dH 2O | 4.3 |
| Add up to | 10 |
Reverse transcription condition: 42 ℃ of 15min; 85 ℃ of 5sec.
The synthetic employed Auele Specific Primer of cDNA is identical with SYBR Green PCR in real time downstream primer.
C, SYBR Green PCR in real time: carry out SYBR Green PCR in real time according to table 5 system.The PCR in real time data are analyzed through ABI 7300 software.The system the primer is directed to 3 ' section non-coding region of JEV.
Table 5
| Component | Volume (μ l) |
| SYBR Ex Taq | 25 |
| Upstream primer SEQ ID NO. 15 | 0.5 |
| Downstream primer SEQ ID NO. 16 | 0.5 |
| |
1 |
| |
4 |
| dH 2O | 19 |
| Add up to | 50 |
95 ℃ of 30 sec; 95 ℃ of 5 sec, 60 ℃ of 31sec, 40 circulations; Add melting curve.
The result is as shown in Figure 7, and pGP-E1, pGP-E2, pGP-NS3, pGP-NS4b can reach 97.3%, 95.7%, 80.9% and 88.5% respectively to the inhibiting rate that JEV duplicates, and shRNA eukaryon expression plasmid promptly of the present invention all reaches more than 80% the inhibiting rate that JEV duplicates.
（5）Western?Blotting。Collect cell to be detected, behind ultrasonic treatment, utilize BCA test kit (PIERCE) to measure and respectively organize total protein concentration; Every group of total protein of getting same amount carries out SDS-PAGE then, through the commentaries on classics film, after the sealing; Add one anti-(mouse-anti JEV E monoclonal antibody), the confidential reference items group is added one anti-(mouse-anti β-actin monoclonal antibody, doctor's moral company); Incubated at room 2hr, PBS washing 3 times adds two anti-(goat anti-mouse iggs then respectively; Doctor's moral company), use the luminous colour developing of ECL (PIERCE) at last.
The result is as shown in Figure 8; Under the situation of each group β-actin amount basically identical; PGP-E1, pGP-E2, pGP-NS3, pGP-NS4b group JEV E protein band obviously are shallower than the pGP-NC group, show that JEV E albumen generation obviously reduces after the effect of shRNA expression plasmid.
3.shRNA the infection of control JEV on the animal level
Get the approaching suckling mouse of body weight (available from model animal institute of Nanjing University), divide 6 groups, be respectively pGP-E1, pGP-E2, pGP-NS3, pGP-NS4b, Virus control and (only attack the poison group; Down with) and Mock (only inject PBS; Group down together), is done immunohistochemical analysis by 3/group; Get the female mouse (model animal institute of Nanjing University) of body weight BALB/c close, age all around, parallel branch 4 organize greatly that (A ~ D), every big group is divided 5 groups (1 ~ 5) again, and 6/group, the E group is done protectiveness and tested for Mock contrast (6/organize).
(1) immunohistochemical methods.With shRNA expression plasmid of the present invention with 100 μ g/ dosage encephalic chamber injection suckling mouse only, behind the 24h with 100LD
50The same encephalic of JEV (NJ2008) dosage chamber injection suckling mouse, get suckling mouse mouse brain behind the 96h and immerse in the formalin solution; According to immunohistochemical methods (LP method) operation steps, make tissue paraffin section de, more respectively with one anti-(mouse-anti JEV E monoclonal antibody), two anti-(HRP-SPA, doctor's moral biotech firms) reaction, DAB develop the color (available from doctor's moral company); Direct viewing in microscope is taken pictures.
Shown in Figure 9; The positive expression site of DAB colour developing back tissue is a brown; Be to exist the JEV particle promptly to dye brown in the animal tissues; PGP-E1, pGP-E2, pGP-NS3, pGP-NS4b group and Mock group dye levels basically identical, and the dyeing of Virus control group is darker show that shRNA expression plasmid of the present invention can effectively prevent and treat the infection of animal body to JEV.
(2) protectiveness test.After the grouping of preceding text animal clustering method, give the interior injection of BALB/c mouse cranial cavity pGP-E1, pGP-E2, pGP-NS3, pGP-NS4b, the PBS of 1 ~ 5 group in A ~ D group respectively; Organize A (1 ~ 5) behind the 24h, inject 20LD respectively in B (1 ~ 5) the mouse cranial cavity
50And 100LD
50The JEV of dosage (NJ2008); Group C (1 ~ 4), D (1 ~ 4) is respectively with pGP-E1, pGP-E2, pGP-NS3, pGP-NS4b and 20LD
50Or 100LD
50The JEV of dosage (NJ2008) is the intracranial injection mouse simultaneously, and C5 and D5 only attack poison; The E group is the Mock group; Observe the animal dead situation every day, observed continuously 15 days, the result sees Figure 10.
Figure 10 A, B result show, attack malicious 20LD
50The JEV of dosage, the mouse survival rate all is higher than 50% after the effect of four groups of shRNA expression plasmids; Attack malicious 100LD
50The JEV of dosage, pGP-E1, pGP-E2, pGP-NS4b group survival rate still are higher than 50%, show the ability that shRNA expression plasmid of the present invention has good preventing JEV to infect.
Figure 10 C, D result show, attack malicious 20LD
50And 100LD
50The JEV of dosage, pGP-E1, pGP-E2, group survival rate all are higher than 50%, and pGP-NS4b group survival rate all reaches 33.3%, shows that shRNA expression plasmid of the present invention has the ability of treating the JEV infection preferably.
< 110>Agricultural University Of Nanjing
< 120>a kind ofly can suppress shRNA and the application thereof that JEV duplicates and infects
<160>?12
<210>?1
<211>?58
<212>?DNA
< 213>artificial sequence
<220>
< 223>shRNA-E1 positive-sense strand template sequence
<400>?1
caccggagtg?gactgaacac?tgattcaaga?gatcagtgtt?cagtccactc?cttttttg 58
<210>?2
<211>?58
<212>?DNA
< 213>artificial sequence
<220>
< 223>shRNA-E1 antisense strand template sequence
<400>?2
gatccaaaaa?aggagtggac?tgaacactga?tctcttgaat?cagtgttcag?tccactcc 58
<210>?3
<211>?59
<212>?DNA
< 213>artificial sequence
<220>
< 223>shRNA-E2 positive-sense strand template sequence
<400>3
caccgacaga?gaactcctca?tggattcaag?agatccatga?ggagttctct?gtttttttg 59
<210>?4
<211>?59
<212>?DNA
< 213>artificial sequence
<220>
< 223>shRNA-E2 antisense strand template sequence
<400>?4
gatccaaaaa?aacagagaac?tcctcatgga?tctcttgaat?ccatgaggag?ttctctgtc 59
<210>?5
<211>?59
<212>?DNA
< 213>artificial sequence
<220>
< 223>shRNA-NS3 positive-sense strand template sequence
<400>5
caccgagatc?atgttagaca?acatttcaag?agaatgttgt?ctaacatgat?ctttttttg 59
<210>?6
<211>?59
<212>?DNA
< 213>artificial sequence
<220>
< 223>shRNA-NS3 antisense strand template sequence
<400>?6
gatccaaaaa?aagatcatgt?tagacaacat?tctcttgaaa?tgttgtctaa?catgatctc 59
<210>?7
<211>?58
<212>?DNA
< 213>artificial sequence
<220>
< 223>shRNA-NS3 positive-sense strand template sequence
<400>7
caccgaatgc?cgttgttgac?ggattcaaga?gatccgtcaa?caacggcatt?cttttttg 58
<210>8
<211>?58
<212>?DNA
< 213>artificial sequence
<220>
< 223>shRNA-NS3 antisense strand template sequence
<400>?8
gatccaaaaa?agaatgccgt?tgttgacgga?tctcttgaat?ccgtcaacaa?cggcattc 58
<210>9
<211>?19
<212>?DNA
< 213>encephalitis b virus (Japanese encephalitis virus)
< 220>the E1 gene order of encephalitis b virus 1553 ~ 1571bp
<223>
<400>?9
ggagtggact?gaacactga 19
<210>10
<211>?19
<212>?DNA
< 213>encephalitis b virus (Japanese encephalitis virus)
< 220>the raq gene sequence of encephalitis b virus 1679 ~ 1697bp
<223>
<400>?10
acagagaact?cctcatgga 19
<210>11
<211>?19
<212>?DNA
< 213>encephalitis b virus (Japanese encephalitis virus)
< 220>the NS3 gene order of encephalitis b virus 6083 ~ 6101bp
<223>
<400>?11
agatcatgtt?agacaacat 19
<210>12
<211>?19
<212>?DNA
< 213>encephalitis b virus (Japanese encephalitis virus)
< 220>the NS4b gene order of encephalitis b virus 7350 ~ 7368bp
<223>
<400>?12
gaatgccgtt?gttgacgga 19
<210>13
<211>?59
<212>?DNA
< 213>artificial sequence
< 220>shNC template positive-sense strand sequence
<223>
<400>?13
caccgttctc?cgaacgtgtc?acgtcaagag?attacgtgac?acgttcggag?aattttttg 59
<210>14
<211>?59
<212>?DNA
< 220>shNC template antisense strand sequence
<223>
<400>?14
gatccaaaaa?attctccgaa?cgtgtcacgt?aatctcttga?cgtgacacgt?tcggagaac 59
<210>15
<211>?20
<212>?DNA
< 220>SYBR Green PCR in real time upstream primer sequence
<223>
<400>?15
ccctcagaac?cgtctcggaa 20
<210>16
<211>?25
<212>?DNA
< 220>SYBR Green PCR in real time downstream primer sequence
<223>
<400>?16
ctattcccag?gtgtcaatat?gctgt 25
Claims (3)
1. one kind can be suppressed the shRNA that encephalitis b virus duplicates and infects; The sequence SEQ ID NO.9 that it is characterized in that guarding with encephalitis b virus genome camber is as its target sequence; The positive-sense strand template sequence of this shRNA is SEQ ID NO.1, and the antisense strand template sequence is SEQ ID NO.2.
2. one kind contains the shRNA eukaryon expression plasmid that right requires 1 described shRNA, it is characterized in that this shRNA eukaryon expression plasmid is that template with the described shRNA of claim 1 is connected the shRNA eukaryon expression plasmid that obtains to encephalitis b virus E gene mRNA sequence with linearizing carrier pGPU6/GFP/Neo.
The described shRNA eukaryon expression plasmid of claim 2 preparation suppress that encephalitis b virus duplicates and/or the medicine that infects in application.
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| CN102352359B (en) * | 2011-10-24 | 2013-06-12 | 石河子大学 | Anti-pig O type foot and mouth disease virus shRNA design and carrier construction method |
| CN102433337B (en) * | 2011-11-22 | 2013-03-13 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种科学研究中心 | Seven synthesized shRNA (short hairpin ribonucleic acid) molecules for inhibiting expression of BMPR-1B (bone morphogenetic protein receptor-1B) genes |
| CN102964434B (en) * | 2012-11-22 | 2014-04-02 | 中国人民解放军军事医学科学院微生物流行病研究所 | Recombinant encephalitis B virus and application thereof |
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Non-Patent Citations (3)
| Title |
|---|
| N. Manjunath et al.Lentiviral delivery of short hairpin RNAs.《Advanced Drug Delivery Reviews》.2009,第61卷第732-745页. * |
| Wen-Bao Qi et al.Effective inhibition of Japanese encephalitis virus replication by small interfering RNAs targeting the NS5 gene.《Virus Research》.2008,第132卷第145-151页. * |
| 吴鹏等.乙脑病毒非结构蛋白NS1的原核表达及重组蛋白间接ELISA方法的初步建立.《畜牧与兽医》.2010,第42卷(第3期),第5-8页. * |
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