CN102206645B - Mediating method of RNAi (ribonucleic acid interference) utilizing lentiviral vector - Google Patents
Mediating method of RNAi (ribonucleic acid interference) utilizing lentiviral vector Download PDFInfo
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Abstract
The present invention relates to a mediating method of RNAi (ribonucleic acid interference) utilizing a lentiviral vector. The method comprises the following specific steps of: firstly carrying out homology analysis on the nucleic acid sequence of relevant viruses to find out conserved regions and designing siRNA (small interfering ribonucleic acid) corresponding to the conserved regions of the related viruses; and cloning the siRNA to an expression plasmid pLVTH, then co-transfecting 293T cells together with pCMV-dR8.91 and pMD2.G, collecting the supernatant of the transfected cells, carrying out ultracentrifugation, purification and concentration to obtain a high-titer lentiviral vector expressing the siRNA, then carrying out cotransduction into target cells together with LV-tTR-KRAB, and strictly controlling the acting time and dose of siRNA drugs utilizing tetracycline analogue DOX. Because of high transduction rate and a stringent control system, the mediating method can actually evaluate the in-vivo antiviral action of the siRNA and can track the whole process of drug interference action, thus having clinical application prospects.
Description
Technical field
The present invention relates to information biology and Molecular Virology technical field, be specifically related to a kind of antiviral method of RNAi research of the lentivirus-mediated based on doxycycline control.
Background technology
A lot of laboratories utilize the siRNA interference to carry out antiviral study in the world in recent years, and a lot of results are conflicting or interference effect is not obvious.Crucial problem is that the working model that they use mostly only limits to use the clone of transfection.Because transfection efficiency is extremely limited, be difficult to the siRNA antiviral effect entered in born of the same parents is really estimated.Afterwards, RNAi usually utilized virus vector to express siRNA, wherein commonly retroviral vector and adenovirus carrier.But retrovirus only infects division cells, the DNA fragmentation that holds foreign gene is no more than 8kb; During the adenovirus carrier cells infected, viral DNA is free in nucleus, can not be incorporated in host chromosome, can not realize stable long-term expression in vivo, and application easily causes immune response repeatedly, more and more be subject to people's attention so derive from the lentiviral vectors (Lentiviral vector) of human immunodeficiency virus-1(HIV-1).
Lentiviral vectors both can infect somatoblast, also can infect Unseparated Cell such as neurocyte, liver cell, hemopoietic stem cell and muscle cell etc., the exogenous genetic fragment shifted is large, host range is very wide, and reduced the chance of restructuring, most important characteristics are that it can be incorporated in host's genome, thus expression alien gene that can high-efficient and lasting, thereby become the carrier that the eukaryotic gene widely paid close attention to shifts.Modified lentiviral vectors later can be realized induction regulating controlling in eukaryotic cell, such as on lentiviral vectors, adding a multiple control switch, just can realize the controlled expression to foreign gene by tsiklomitsin or tetracycline analogue.
The structure of lentiviral vectors is very simple, and principle is exactly that the cis-acting elements in the HIV-1 genome (as packaging signal, long terminal repeat) is separated with the sequence of coding trans-acting albumen.Build at present slow virus two plasmid expression systems, three-plasmid system and four plasmid expression systems are arranged.The most frequently used is three-plasmid system (packaging plasmid, envelope protein plasmid and transferring plasmid): packaging plasmid is used for expressing HIV-1 and copies required whole trans-activators, but does not produce viral envelope protein and accessory protein vpu; The plasmid-encoded vesicular stomatitis virus G of envelope protein albumen (VSV-G); In transferring plasmid, contain the H1 promotor, can be in host cell the continuous expression foreign gene, express simultaneously the Factor1 by EF1(Elongation) green fluorescent protein (GFP) of promoters driven, transfection efficiency and infect the detection of the efficiency of infection of purpose cell while packing for virus.The sharpest edges of three-plasmid system are that the overlapping chance of sequence is reduced greatly, thereby have reduced the possibility that produces RCV in the carrier regrouping process.
The slow virus carrier system that has the multiple control switch, can make transfection efficiency increase substantially more than 90%.For this reason, we will utilize this system deeply to estimate the antiviral effect of siRNA.Due to high transfection efficiency, will make the antiviral effect of medicine obtain estimating more really, also more likely for the special target medicine in body, discharge in the future.In addition, the tsiklomitsin switch that this system has, can not only make the interference effect of investigator's probe siRNA medicine, and by on-off control, can follow the trail of the process of drug intervention, provides possibility for exploring pharmacokinetics, metabolism and toxicology.Therefore, set up this system and will provide a very practical instrument for antiviral research.
Summary of the invention
Technical problem to be solved by this invention is: the RNAi technology of using the lentivirus-mediated of doxycycline control, provide a kind of siRNA of utilization research antiviral method, so that the interference effect to the siRNA medicine is carried out detailed assessment, and follow the trail of pharmaceutically-active whole process by on-off control, for the research of antiviral is made contributions.
The present invention solves its technical problem and adopts following technical scheme:
The present invention is to provide the method for lentivirus-mediated RNAi based on doxycycline control a kind of, specifically: at first, the biological software that utilization is correlated with is carried out homology analysis to the nucleotide sequence of correlated virus, finds out conservative region, designs the siRNA for the correlated virus conservative region; Again synthetic siRNA is cloned in expression plasmid pLVTH, then by this expression plasmid and the common transfection 293T of pCMV-dR8.91, pMD2.G cell, cell conditioned medium after the collection transfection, through ultracentrifugation, purifying, after concentrated, formed the lentiviral vectors of expressing the high titre of siRNA, the lentiviral vectors that finally will express siRNA imports target cell with the lentiviral vectors LV-tTR-KRAB corotation of controlling the tsiklomitsin switch, and utilizes tetracycline analogue DOX strictly to control the pharmaceutically-active Time and dosages of siRNA.
The present invention can adopt the method comprised the following steps:
(1) screening siRNAs:
First gene order U95551 according to HBV in GeneBank, principle with reference to the siRNA design, length in siRNA design website http://rnadesigner.classic.invitrogen.com/rnaiexpress/index.jsp Select gene coding region AA beginning is 21nt, the GC% conservative nucleotide sequence between 45%-55%, avoid the non-coding region of 5 ' and 3 ' end, again the sequence of selection is carried out to the analysis of BLAST homology, avoid existing with human genome the interference sequence of homology.
(2) to the evaluation of the siRNAs that filters out:
Different siRNAs is building up in carrier for expression of eukaryon pSuper, and whether these several siRNAs are influential to virus for preliminary assessment, and its method is: utilize Lipofectamine2000
TM(purchased from American I nvitrogen company) advances the pSuper-siRNA transfection in cell, and cell conditioned medium and cell after the collection transfection utilize Protocols in Molecular Biology to detect the variation of corresponding viral index;
(3) lentiviral vectors of construction expression siRNA:
Respectively these several siRNAs are cloned in lentivirus transfer plasmid pLVTH, the transferring plasmid pLVTH-HBV-siRNA that then will build and packaging plasmid pCMV-dr8.91, coating plasmid pMD2.G advances to converge in the 293T cell that rate is 80% left and right with calcium phosphate transfection test kit (purchased from green skies company) cotransfection according to the ratio of every 60mm Tissue Culture Dish 15 μ g:10 μ g:5 μ g, collect the cell conditioned medium of the rear 48h of transduction, the centrifugal 5-10min of 2500rpm, supernatant is removed to cell debris with 0.45 μ m membrane filtration, then the centrifugal 2hours of 24500rpm, supernatant discarded, finally use Opti-MEM(purchased from U.S. Gibco company) resuspended virus,
(4) survey the titre of lentiviral vectors:
According to 10 times of gradient dilutions, each that then adds respectively 96 orifice plates is aerial, cultivates to incubator by resuspended virus; After overnight incubation, renew bright cell culture fluid and continue to cultivate; After 48h, observe fluorescence, number goes out the number of cell in the hole that fluorocyte accounts for 9-11%; According to following formula, calculate virus titer again:
Tilter (/ml)=10%GFP
+Cells * total cell count * viral dilution multiple,
The RNAi of the lentivirus-mediated (5) estimate built is on viral impact:
The lentiviral vectors built is transduceed into target cell according to the titre of MOI=10, transduction can add the Polybrene(of 6 μ g/mL simultaneously purchased from U.S. Sigma company) to improve transduction efficiency, the then variation of different time viral replication in and expression level after the detailed assessment transduction;
(6) build the RNAi system of the lentivirus-mediated of doxycycline control:
Expression plasmid and slow virus packaging plasmid pCMV-dr8.91, the coating plasmid pMD2.G cotransduction of expressing tsiklomitsin arrestin tTR-KRAB are advanced in the 293T cell, the centrifugal 5-10min of cell conditioned medium 2500rpm after collection 48h, by supernatant with 0.45 μ m membrane filtration, then the centrifugal 2hours of 24500rpm, supernatant discarded, finally use the resuspended virus of Opti-MEM, make viral titre reach consistent with the titre of the lentiviral vectors of expressing siRNA;
(7) observe the impact of the RNAi of the lentivirus-mediated that DOX induces on virus replication and expression: the slow virus that will express siRNA advances in target cell with the slow virus linked transduction of expression tsiklomitsin modulin tTR-KRAB, second day is divided into 2 groups by cell, one group adds DOX and processes, another group does not add DOX and processes, after 4 days, collect respectively this two groups of cells, the nucleocapsid of the intracellular HBV of extracting, half directly is used for being Western blotting, and purpose is to detect the variation of HBV core protein expression level; Second half by the HBV DNA extracting and purifying in nucleocapsid out, uses P
32The probe of mark is Southern blotting and is detected, and its objective is the situation that copies of observing HBV in born of the same parents.
The present invention can adopt following methods screening siRNAs:
First gene order U95551 according to HBV in GeneBank, principle with reference to the siRNA design, length in siRNA design website http://rnadesigner.classic.invitrogen.com/rnaiexpress/index.jsp Select gene coding region AA beginning is 21nt, the GC% conservative nucleotide sequence between 45%-55%, avoid the non-coding region of 5 ' and 3 ' end, again the sequence of selection is carried out to the analysis of BLAST homology, avoid existing with human genome the interference sequence of homology; Then according to the requirement of pSuper carrier, the two ends of design 60-64nt comprise respectively designs an irrelevant interference sequence simultaneously as negative control by the oligonucleotide chain of the restriction enzyme site of Bgl II and Hind III.
The described method of utilizing Protocols in Molecular Biology to detect the variation of corresponding viral index is: utilize and comprise that the Protocols in Molecular Biology of ELISA, real-time PCR, RT-PCR, Western blotting, Southern blotting detects respectively in the copy number of viral HBsAg and HBeAg, supernatant HBV, born of the same parents the variation of hbv replication level in the variation of HBV core protein expression level in HBV mrna expression situation, born of the same parents and born of the same parents.
Described method by the resuspended virus of Opti-MEM is: lentiviral vectors is with after 24500rpm ultracentrifugation 2hours, the resuspended virus of Opti-MEM with the ice of 100 μ l, the advantage that adopts Opti-MEM is the condition in the time of can optimizing the virus vector transduction, improves the level of virus transduction.In by the resuspended virus of Opti-MEM, virus is placed on ice to 2hours at least, and operation gently, can not produce bubble.
The present invention can adopt the level of following methods detailed assessment virus replication and expression:
Lentiviral vectors is transduceed after target cell, at following different time point: and collecting cell supernatant when 24h, 48h, 72h, 96h and 1week, and then collect the cell after 1week, for detection of the variation of different viral indexs.In cell conditioned medium, the variation of the HBsAg of HBV and HBeAg level is to detect with section's China ELISA test kit; In supernatant, the variation of viral copy number is to utilize real-time PCR to detect, and wherein the extracting of supernatant HBV DNA is extracted the operation of test kit operation instructions according to TIANGEN virus genom DNA/RNA, finally in Aligent Technology software, analyzes; The variation of born of the same parents' inner virus rna level is to adopt real-time fluorescent RT-PCR method for detecting (using cell house-keeping gene β-actin as reference gene), then in Aligent Technology software, analyzes; And the variation of the levels of replication of born of the same parents' inner virus is to utilize P
32The Southern blotting of mark detects the content of HBV DNA in the intracellular nucleic capsid; The variation of the expression level of born of the same parents' inner virus albumen is to utilize Western blotting to detect in born of the same parents the content of core albumen in nucleocapsid, and in born of the same parents GAPDH content as internal reference.
The present invention can adopt following methods to observe the variation of virus replication and expression level in the situation that DOX adds or DOX does not add: the slow virus that will express siRNA advances target cell with the slow virus linked transduction of expression tsiklomitsin modulin tTR-KRAB, second day is divided into 2 groups by cell, one group adds DOX and processes, another group does not add DOX and processes, after 4 days, collect respectively this two groups of cells, the nucleocapsid of the intracellular HBV of extracting, half directly is used for being Western blotting, and purpose is to detect the variation of HBV core protein expression level; Second half by the HBV DNA extracting and purifying in nucleocapsid out, uses P
32The probe of mark is Southern blotting and is detected, and its objective is the situation that copies of observing HBV in born of the same parents.
The present invention has following main technical superiority and effect:
One. utilize bioinformatics technique to design three couples of siRNAs for the hepatitis B viruses (HBV) conserved regions, respectively HBVDe DR1 district (1826nt-1845nt), CP district (1775nt-1795nt) and RT district (672nt-692nt), siRNAs is building up to respectively in lentiviral vectors by synthetic this is several, transduces in hepatoma cell line HepG2.2.15.The variation of HBsAg, HBeAg and HBV virus copy number in cell conditioned medium while then detecting 24h, 48h after transduction, 72h, 96h, 1week, and the intracellular virus situation that copies and express.The RNAi of the lentivirus-mediated of discovery under controlling based on doxycycline not only strongly inhibited hbv replication and expression, and this restraining effect is the strict control that is subject to doxycycline.This provides very important using value for antiviral therapy.
They are two years old. and utilize the RNAi technology of the lentivirus-mediated that a kind of novel doxycycline controls, set up a kind of antiviral novel method.Because this carrier has efficiently, safety, high degree of specificity, can effectively control copying and expression of virus, thereby for controlling virus infection and the treatment virus disease is made contributions.
They are three years old. and in view of the siRNA expression vector of current use such as carrier for expression of eukaryon or adenovirus carrier have defect separately, based on above-mentioned effectiveness, the present invention also has following advantage and effect:
At first the RNAi that utilizes doxycycline to control lentivirus-mediated has solved the low difficult problem of siRNA transfer efficiency, utilizes lentiviral vectors siRNA can be transduceed efficiently into various cells, comprises somatoblast and Unseparated Cell; Secondly lentiviral vectors can be incorporated in the genome of target cell, has realized the continuous expression to siRNA, and the RNAi that the present invention detects lentivirus-mediated can maintain the time in 1 week to the strong restraining effect of HBV; After the most important thing is that the present invention shows and adds DOX, the RNAi of lentivirus-mediated is the strongly inhibited hbv replication not only, and to cell injury not, and in case remove DOX, this interference effect disappears immediately.This shows that this interference effect is the strict control that is subject to doxycycline, this has more security than other carriers, the cellulotoxic side effect of having avoided continuous expression external source siRNA and may having caused, simultaneously can pharmaceutically-active time of strict control siRNA and dosage by the DOX switch, more help the research of pharmacokinetics; And the lentiviral vectors that utilizes three-plasmid system (expression plasmid, coating plasmid, transferring plasmid) to build, with other virus vector, to compare, its structure is more simple and convenient, more can not produce recombinant virus.Therefore, this invention has the characteristics such as safety, efficient, cost is low, is suitable for very much antiviral treatment research.This also provides more wide prospect for developing the more effective method of antiviral therapy.
The accompanying drawing explanation
Fig. 1 is the affect schematic diagram of the RNAi of lentivirus mediated on HBsAg secretion level in cell conditioned medium.
Fig. 2 is the affect schematic diagram of the RNAi of lentivirus mediated on HBeAg secretion level in cell conditioned medium.
Fig. 3 is the affect schematic diagram of the RNAi of lentivirus mediated on HBV mRNA level in cell.
Fig. 4 is the regulating effect schematic diagram of the RNAi of the DOX lentivirus mediated of inducing to the HBVCore protein expression.
Fig. 5 is the regulating effect schematic diagram of the RNAi of the DOX lentivirus mediated of inducing to hbv replication.
Fig. 6 is the packing schematic diagram of the DOX lentiviral vectors of inducing.
Embodiment
The invention will be further described below in conjunction with example and accompanying drawing:
The method of utilizing the slow virus carrier system mediate rna i of doxycycline control provided by the invention comprises the following steps:
1. utilize information biology to filter out the siRNAs for the different conserved regions of HBV, first gene order U95551 according to HBV in GeneBank, principle with reference to the siRNA design, length in siRNA design website http://rnadesigner.classic.invitrogen.com/rnaiexpress/index.jsp Select gene coding region AA beginning is 21nt, the GC% conservative nucleotide sequence between 45%-55%, avoid the non-coding region of 5 ' and 3 ' end, again the sequence of selection is carried out to the analysis of BLAST homology, avoid existing with human genome the interference sequence of homology; Then according to the requirement of pSuper carrier, the two ends of design 60-64nt comprise that respectively Bgl II and Hind III(are purchased from Takara company) the oligonucleotide chain of restriction enzyme site, design simultaneously an irrelevant interference sequence as negative control, its process, in Table 1, is then synthesized the interference sequence designed.(Invitrogen company is synthetic)
2. different siRNAs is building up in carrier for expression of eukaryon pSuper, whether this several influential to HBV to siRNAs for preliminary assessment: carry the day before yesterday by hepatoma cell line HepG2.2.15 cell that can continuous expression HBV according to 5 * 10
5The density in/hole is inoculated in 6 orifice plates, and during transfection, the cytogamy degree will reach 80-90%.Second day utilizes Lipofectamine2000
TMBy pSuper-siRNA(according to plasmid: Lipofectamine2000
TMThe ratio of=4 μ g:10 μ l) transfection is advanced in the HepG2.2.15 cell, after 6 hours, renewing the bright cell culture fluid that contains 10%FBS continues to cultivate, then collect cell conditioned medium and the cell of different time points after transfection, the ELISA(ELISA test kit is purchased from Shanghai Xiamen Kehua), RT-PCR and real-time PCR(relevant primer synthetic by Invitrogen company) detect respectively the variation of HBsAg and HBeAg, HBV mRNA and HBV copy number.Result shows that these disturb site, to hbv replication and expression, certain restraining effect is arranged.
3. the lentiviral vectors of construction expression siRNA: respectively these several siRNAs are cloned in lentivirus transfer plasmid pLVTH, the transferring plasmid pLVTH-HBV-siRNA that then will build and packaging plasmid pCMV-dr8.91, coating plasmid pMD2.G advances in the 293T cell with calcium phosphate transfection test kit (purchased from green skies company) cotransfection according to the ratio of every 60mm Tissue Culture Dish 15 μ g:10 μ g:5 μ g, collect the cell conditioned medium of the rear 48h of transduction, the centrifugal 5-10min of 2500rpm, by supernatant with 0.45 μ m membrane filtration, then use ultracentrifuge (Beckman company) at 24500rpm, 4 ℃ of ultracentrifugation 2hours, careful supernatant discarded, ice bath at least 2 hours, use finally that Opti-MEM is resuspended and packing is viral.
4. survey the titre of lentiviral vectors: measure the day before yesterday by the HepG2.2.15 cell with 5 * 10
4The density in/hole is inoculated in 96 orifice plates.Prepare 6-9 aseptic EP pipe, add the fresh medium of 90 μ l.Virus stock solution used to be determined 10 μ l are added in first EP pipe, mix, get 10 μ l and join in second EP pipe, by that analogy until last pipe.By the original cell culture fluid of sucking-off in selected cell hole, the virus liquid diluted is added respectively in each hole, as in incubator, cultivating.After overnight incubation, renew bright cell culture fluid 100 μ l, continue to cultivate.After 48h, observe fluorescence.Number goes out the number of cell in the hole that fluorocyte accounts for 10% left and right.According to following formula, calculate virus titer:
Tilter (/ml)=%GFP
+Cells * total cell count * viral dilution multiple.
5. the impact of the RNAi of the lentivirus-mediated estimate built on hbv replication and expression: carry the day before yesterday by the HepG2.2.15 cell with 5 * 10
5the density in/hole is inoculated in 6 orifice plates, in the titre transduction HepG2.2.15 cell of the lentiviral vectors that second day will build according to MOI=10, transduction can add the Polybrene(of 6 μ g/mL simultaneously purchased from U.S. Sigma company) to improve transduction efficiency, after virus is hatched 12h, renewing the bright nutrient solution that contains 10%FBS continues to cultivate, then collect the cell conditioned medium of the rear different time points of transduction, last collecting cell, utilize ELISA, RT-PCR, real-time PCR, Western blotting and Southern blotting detect respectively HBsAg and the HBeAg of HBV, HBV mRNA, viral loads, the situation of HBV core albumen and hbv replication.
6. build the RNAi system of the lentivirus-mediated of doxycycline control: expression plasmid and the slow virus packaging plasmid pCMV-dr8.91 that will express tsiklomitsin arrestin tTR-KRAB, coating plasmid pMD2.G advances in the 293T cell with calcium phosphate transfection test kit (purchased from green skies company) cotransfection according to the ratio of every 60mm Tissue Culture Dish 15 μ g:10 μ g:5 μ g, after 8 hours, renewing the bright nutrient solution that contains 10%FBS continues to cultivate, cell conditioned medium after collection 48h, the centrifugal 5-10min of 2500rpm, by supernatant with 0.45 μ m membrane filtration, then use ultracentrifuge (Beckman company) at 24500rpm, 4 ℃ of ultracentrifugation 2hours, careful supernatant discarded, ice bath at least 2 hours, use finally that Opti-MEM is resuspended and packing is viral, make viral titre reach consistent with the titre of the lentiviral vectors of expressing siRNA.
7. the slow virus that will express siRNA advances in target cell with the slow virus linked transduction of expressing tsiklomitsin modulin tTR-KRAB, observes then that DOX adds or do not add the impact on hbv replication and expression: carry the day before yesterday by the HepG2.2.15 cell with 5 * 10
5The density in/hole is inoculated in 6 orifice plates, second day is just expressed the slow virus of siRNA and all with the titre of MOI=10, is transduceed into the HepG2.2.15 cell with the slow virus of expressing tsiklomitsin modulin tTR-KRAB, add simultaneously the Polybrene(of 6 μ g/mL purchased from U.S. Sigma company), after virus is hatched 12h, cell is divided into two, and half adds and contains 5 μ g/mLDOX(purchased from U.S. Sigma company) fresh medium; Second half adds the fresh medium that does not contain DOX, continues to cultivate; Collecting cell after 4 days, the HBV nucleocapsid in the extracting born of the same parents, half is used for doing the variation that Western blotting detects HBV Core protein expression level, and the HBV DNA that second half extracts in nucleocapsid, then use P
32The probe of mark is Southern blotting to detect the variation of HBV DNA replication dna level.
8. interpretation of result:
(1) siRNA for the different conserved regions of HBV of lentivirus mediated has inhibition in various degree to HBV, referring to Fig. 1, Fig. 2, Fig. 3 and table 2.Presentation of results is as follows:
Each sample is got the mean value of three experiments, HBeAg and HBsAg value in untreated fish group HepG2.2.15 cell conditioned medium are made as to 100, and visible LV-HBV-siRNA2 (1775-1795i) and LV-HBV-siRNA3 (1826-1845i) have significant restraining effect on the level of HBsAg and HBeAg.Wherein, during 72h, LV-HBV-siRNA3 (1826-1845i) reaches maximum to the inhibition of HBsAg after transduction, and inhibiting rate is 98.5%; Corresponding LV-HBV-siRNA2 (1775-1795i) reaches 90.9% to the inhibition of HBsAg.It is upper that the trend of this inhibition is embodied in HBeAg equally: during 96h, LV-HBV-siRNA3 (1826-1845i) reaches maximum to the inhibition of HBeAg after transduction, and inhibiting rate is 73.5%; LV-HBV-siRNA2 (1775-1795i) reaches 44.9%(such as Fig. 1 and Fig. 2 to the inhibiting rate of HBeAg).
After transduction during 96h, LV-HBV-siRNA3 has reduced more than 10 times the DNA copy number of HBV in supernatant, and LV-HBV-siRNA2 can reduce approximately 9 times by the DNA copy number of HBV in supernatant, the LV-HBV-siRNA1 of suppression efficiency minimum also can reduce by 4 times of left and right (table 2) by the DNA copy number of HBV.
The variation of HBV mRNA level is that amount according to HBV Auele Specific Primer level measures out divided by reference gene β-actin in born of the same parents.Each sample is to get average.The ratio of untreated fish group is made as to 100, and ratio is greater than 100 and means that HBV mRNA level is higher than untreated fish group, and namely the mRNA level rises; On the contrary, if ratio is less than 100, mean that HBV mRNA level reduces, be worth less expression interference effect better.Result shows: with untreated fish group, compare, LV-HBV-siRNA3(1826-1845i) the mRNA level of HBV can be reduced to 98.1%; And LV-HBV-siRNA2(1775-1795i) make HBV mRNA level reduce by 82.8% (Fig. 3); This ELISA and real time PCR result same and front are consistent, and LV-HBV-siRNA3(1826-1845i is described) and LV-HBV-siRNA2(1775-1795i) transcription product of HBV is had to very strong restraining effect equally.
(2) slow virus carrier system that utilizes DOX to control not only can be controlled copying and expressing of HBV strongly, and this restraining effect is (Fig. 4 and the Fig. 5) that is subject to the strict control of DOX.
Fig. 4 does not have in the situation of DOX, even LV-HBV-siRNA3 and LV-tTR-KRAB infect simultaneously, the core albumen of the HBV normal expression that remains unchanged, but in case add DOX(5 μ g mL-1), the RNAi of lentivirus mediated demonstrates very strong interference effect immediately, and the expression amount of the core albumen of HBV descends immediately.And, iff being that LV-HBV-siRNA3 infects the HepG2.2.15 cell, no matter whether the existence of DOX is arranged, all show strong inhibition.And the expression level of internal reference GAPDH without any variation.
Fig. 5 does not have in the situation of DOX, LV-HBV-siRNA3 and LV-tTR-KRAB infect simultaneously, copying of HBV still normally carried out, but in case add DOX(5 μ g mL-1), the RNAi of lentivirus mediated demonstrates very strong interference effect immediately, copying of HBV is suppressed, the content of RC-DNA, DL-DNA and ssDNA all significantly reduces, and iff being LV-HBV-siRNA3 infection HepG2.2.15 cell, no matter whether the existence of DOX is arranged, all shown strong inhibition, this result with Western blotting is consistent.HBV completes its reproduction process in nucleocapsid in born of the same parents, the replicative intermediate of HBV comprises that in a single day the content of RC-DNA, DL-DNA and ssDNA be lowered, and its process copied just can't complete.
The present embodiment provides the packing schematic diagram of the lentiviral vectors that DOX as shown in Figure 6 induces, and its process is: the lentiviral vectors LV-siRNA of construction expression siRNA and express the lentiviral vectors LV-tTR-KRAB of tsiklomitsin arrestin in the 293T cell at first, its process is to utilize calcium phosphate transfection test kit (purchased from green skies company) to join respectively in the cell ware (purchased from U.S. Corning company) of the 60mm that has inoculated the 293T cell the day before yesterday according to the ratio of pLVTH-siRNA:pCMV-dr8.91:pMD2.G=15 μ g:10 μ g:5 μ g and pLV-tTR-KRAB:pCMV-dr8.91:pMD2.G=15 μ g:10 μ g:5 μ g, then collect respectively the cell conditioned medium of 48h after transfection, the centrifugal 5-10min of 2500rpm, by supernatant with 0.45 μ m membrane filtration, then use ultracentrifuge (Beckman company) at 24500rpm, 4 ℃ of ultracentrifugation 2hours, careful supernatant discarded, ice bath at least 2 hours, finally use the resuspended virus of Opti-MEM, secondly, these two virus vector are transduceed in target cell with same virus titer, in a single day lentiviral vectors enters target cell, its genome can be incorporated on host's genome, yet, after the lentiviral vectors of expression tsiklomitsin arrestin enters target cell, express tTR-KRAB albumen, this albumen is upper by the tetO that the DNA binding domains is attached to the lentiviral vectors of expressing siRNA, thereby closes the expression of genes involved, now, if add tetracycline analogue doxycycline (DOX), DOX can be attached on tTR-KRAB albumen at once, change its structural domain, thereby make it can not be in conjunction with tetO, and then the expression of opening the relevant interference gene, siRNA enters in tenuigenin after in nucleus, transcribing, then by the Dicer enzyme in tenuigenin, cut into the siRNA of 19-21nt, then the albumen polymerization shape RNA silencing complex (RISC) in siRNA and born of the same parents, the said target mrna of homology with it of finally degrading, reach its restraining effect.
Subordinate list
Table 1 utilizes information biology to filter out the siRNAs for the different conserved regions of HBV
The impact of the RNAi of table 2 lentivirus mediated on HBV virion secretion level in cell conditioned medium
Time（HBV?DNA?copy×10
4mL
-1）
Sequence table
<110 > Wuhan Virology Institute,Chinan academy of Sciences
<120 > utilize the method for lentivirus-mediated RNAi
<140>
<141>
<160>1
<170>
<210>1
<211>21
<212>DNA
<213 > for the target sequence of the siRNA (N) of HBV conserved regions
<220>
<221>
<222>
<223>
<400>1
CTAGTGAAGGGCGTATGATTA
<210>2
<211>19
<212>DNA
<213 > for the target sequence of the siRNA1 of HBV conserved regions
<220>
<221>
<222>
<223>
<400>2
GCTCAGTTTACTAGTGCCA
<210>3
<211>21
<212>DNA
<213 > for the target sequence of the siRNA2 of HBV conserved regions
<220>
<221>
<222>
<223>
<400>3
CTAGGAGGCTGTAGGCATAAA
<210>4
<211>19
<212>DNA
<213 > for the target sequence of the siRNA3 of HBV conserved regions
<220>
<221>
<222>
<223>
<400>4
TTCACCTCTGCCTAATCAT
Claims (7)
1. method of utilizing lentivirus-mediated RNAi, a kind of method that it is characterized in that lentivirus-mediated RNAi based on doxycycline control, specifically: at first, the biological software that utilization is correlated with is carried out homology analysis to the nucleotide sequence of hepatitis b virus hbv, find out conservative region, design the listed siRNA for the hepatitis b virus hbv conservative region of following table:
Again synthetic siRNA is cloned in expression plasmid pLVTH,
Then by this expression plasmid and the common transfection 293T of pCMV-dR8.91, pMD2.G cell, collect cell conditioned medium after transfection, through ultracentrifugation, purifying, after concentrated, formed the lentiviral vectors of expressing the high titre of siRNA,
The lentiviral vectors that finally will express siRNA imports in the HepG2.2.15 cell with the lentiviral vectors LV-tTR-KRAB corotation of controlling the tsiklomitsin switch, and utilizes doxycycline strictly to control the pharmaceutically-active Time and dosages of siRNA.
2. method according to claim 1 is characterized in that adopting the method comprised the following steps:
(1) screening siRNAs:
Utilize information biology to filter out several siRNAs for corresponding virus: first gene order according to hepatitis b virus hbv in GeneBank, principle with reference to the siRNA design, the length of Select gene coding region AA beginning is 21nt, the GC% conservative nucleotide sequence between 45%-55%, avoid the non-coding region of 5 ' and 3 ' end, again the sequence of selection is carried out to the analysis of BLAST homology, avoid existing with human genome the interference sequence of homology;
(2) to the evaluation of the siRNAs that filters out:
Different siRNAs is building up in carrier for expression of eukaryon pSuper, and whether these several siRNAs are influential to virus for preliminary assessment, and its method is: utilize Lipofectamine2000
TMThe pSuper-siRNA transfection is advanced in cell, and cell conditioned medium and cell after the collection transfection, utilize Protocols in Molecular Biology to detect the variation of corresponding viral index;
(3) lentiviral vectors of construction expression siRNA:
Respectively these several siRNAs are cloned in lentivirus transfer plasmid pLVTH, the transferring plasmid pLVTH-HBV-siRNA that then will build and packaging plasmid pCMV-dr8.91, coating plasmid pMD2.G cotransduction are advanced in the 293T cell, collect the cell conditioned medium of the rear 48h of transduction, the centrifugal 5-10min of 2500rpm, by supernatant with 0.45 μ m membrane filtration, then the centrifugal 2hours of 24500rpm, supernatant discarded, finally use the resuspended virus of Opti-MEM;
(4) survey the titre of lentiviral vectors:
Resuspended virus, according to 10 times of dilutions, is then added respectively in each hole, cultivate to incubator; After overnight incubation, renew bright cell culture fluid and continue to cultivate; After 48h, observe fluorescence, number goes out the number of cell in the hole that fluorocyte accounts for 9-11%; According to following formula, calculate virus titer again:
Tilter (/ml)=10%GFP
+Cells * total cell count * viral dilution multiple,
The RNAi of the lentivirus-mediated (5) estimate built is on viral impact:
The lentiviral vectors built is transduceed into cell according to the titre of MOI=10, then the level of detailed assessment virus replication and expression;
(6) build the RNAi system of the lentivirus-mediated of doxycycline control:
Expression plasmid and slow virus packaging plasmid pCMV-dr8.91, the coating plasmid pMD2.G cotransduction of expressing tsiklomitsin arrestin tTR-KRAB are advanced in the 293T cell, cell conditioned medium after collection 48h, the centrifugal 5-10min of 2500rpm, by supernatant with 0.45 μ m membrane filtration, then the centrifugal 2hours of 24500rpm, supernatant discarded, finally use the resuspended virus of Opti-MEM, makes viral titre reach consistent with the titre of the lentiviral vectors of expressing siRNA;
(7) slow virus that will express siRNA advances in target cell with the slow virus linked transduction of expressing tsiklomitsin modulin tTR-KRAB, then observes the variation of virus replication and expression level in the situation that doxycycline adds or do not add.
3. method according to claim 2 is characterized in that adopting following methods screening siRNAs:
First gene order U95551 according to HBV in GeneBank, principle with reference to the siRNA design, the length of Select gene coding region AA beginning is 21nt, the GC% conservative nucleotide sequence between 45%-55%, avoid the non-coding region of 5 ' and 3 ' end, again the sequence of selection is carried out to the analysis of BLAST homology, avoid existing with human genome the interference sequence of homology; Then according to the requirement of pSuper carrier, the two ends of design 60-64nt comprise respectively designs an irrelevant interference sequence simultaneously as negative control by the oligonucleotide chain of the restriction enzyme site of Bgl II and Hind III.
4. method according to claim 2 is characterized in that the method for utilizing Protocols in Molecular Biology to detect the variation of corresponding viral index is:
Utilization comprises that the Protocols in Molecular Biology of ELISA, real-time PCR, RT-PCR, Western blotting, Southern blotting detects respectively in the copy number of viral HBsAg and HBeAg, supernatant HBV, born of the same parents the variation of hbv replication level in the variation of HBV core protein expression level in HBV mrna expression situation, born of the same parents and born of the same parents.
5. method according to claim 2 is characterized in that by the method for the resuspended virus of Opti-MEM being:
Lentiviral vectors is with after 24500rpm ultracentrifugation 2hours, and the resuspended slow virus of Opti-MEM with the ice of 100 μ l, now will be placed in slow virus on ice 2h at least, and operation gently, can not produce bubble.
6. method according to claim 2 is characterized in that adopting the level of following methods detailed assessment virus replication and expression:
Lentiviral vectors is transduceed after the HepG2.2.15 cell, at the time point of 24h, 48h, 72h, 96h and 1week collecting cell supernatant respectively, then collects the cell after 1week, for detection of the variation of different viral indexs; In cell conditioned medium, the variation of the HBsAg of HBV and HBeAg level is to detect with section's China ELISA test kit; In cell conditioned medium, the variation of viral copy number is to utilize real-time PCR to detect, wherein the extracting of supernatant HBV DNA is extracted the operation of test kit operation instructions according to TIANGEN virus genom DNA/RNA, finally in Aligent Technology software, analyzes; The variation of intracellular virus rna level is to adopt real-time fluorescent RT-PCR method for detecting, using cell house-keeping gene β-actin as reference gene, then in Aligent Technology software, analyzes during detection; And the variation of the levels of replication of intracellular virus is to utilize P
32The probe of mark is done the content that Southern blotting detects HBVDNA in the intracellular nucleic capsid; The variation of the expression level of intracellular virus albumen is to utilize Western blotting to detect in born of the same parents the content of core albumen in nucleocapsid, and in cell GAPDH content as internal reference.
7. method according to claim 1 is characterized in that adopting following methods to observe the variation of virus replication and expression level in the situation that doxycycline adds or doxycycline does not add:
The slow virus of expressing siRNA is advanced to target cell with the slow virus linked transduction of expressing tsiklomitsin modulin tTR-KRAB, second day is divided into 2 groups by cell, one group adds powerful mycin and processes, another group does not add powerful mycin and processes, after 4 days, collect respectively this two groups of cells, the nucleocapsid of the intracellular HBV of extracting, half directly is used for being Western blotting, and purpose is to detect the variation of HBV core protein expression level; Second half by the HBV DNA extracting and purifying in nucleocapsid out, uses P
32The probe of mark is Southern blotting and is detected, and its objective is the situation that copies of observing HBV in born of the same parents.
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