CN101906417A - shRNA for hepatitis B virus and recombinant adeno-associated virus vector treating vector carrying same - Google Patents
shRNA for hepatitis B virus and recombinant adeno-associated virus vector treating vector carrying same Download PDFInfo
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Abstract
The invention belongs to the field of biomedicine, and relates to a recombinant adeno-associated virus carrying shRNA (shorthairpin RNA, shRNA) with the effect of inhibiting hepatitis B virus, a preparation method thereof and application thereof in preparing medicaments for treating and/or preventing viral diseases. By adopting gene recombination technology, the shRNA with the effect of inhibiting the hepatitis B virus is cloned into skeleton plasmid of an adeno-associated virus vector, and the skeleton plasmid, together with helper plasmid, performs cotransfection on packaging cells to obtain the recombinant adeno-associated virus. The recombinant adeno-associated virus can effectively inhibit the copy and expression of the hepatitis B virus, and has obvious in-vivo inhibition effect, long lasting time and smaller cytotoxicity.
Description
Technical field
The present invention relates to molecular biology and biological medicine technology field, specifically, the present invention relates to shRNA at hepatitis B virus, carry its carrier and they treat and/or prevent application in the hepatitis B virus infection disease in preparation.
Background technology
The whole world has and surpasses 3.5 hundred million hepatitis B virus (annual nearly 1,000,000 people die from by HBV and infect caused hepatic diseases for hepatitis B virus, HBV) carrier.Chronic HBV infection has become the worldwide disease of serious harm human health.Chronic infectious patients uses Interferon, rabbit and nucleic acid analog to treat usually.In clinical application, because the result of treatment of Interferon, rabbit is undesirable, the highest rate of should beating with a stick has only 40%, and the appearance of the side effect of nucleic acid analog and the problems such as resistance that produced by the HBV sudden change, still need explore medicine and the method for the anti-HBV of novel and effective.
RNA disturbs (RNA interference, RNAi) be by small molecules interference RNA fragment (small interfering RNA, siRNA) thus induce the natural process that causes gene silencing with the degraded of the specificity of its homologous mRNA, be sequence-specific PTGS (post transcriptional gene silencing, PTGS).The RNAi phenomenon is Fire etc.
[1]When research nematode gene function, find, find that thereafter this phenomenon almost is present in all eukaryotic cells.That the performance main effects is the siRNA of the about 21-23bp of a segment length among the RNAi.SiRNA is double-stranded RNA (the double stranded RNA that produces by variety of way by in the specific nucleic acid restriction endonuclease cutting body, dsRNA) form, siRNA unwinds and becomes strand, wherein antisense strand siRNA and corresponding proteins in conjunction with formation induce reticent mixture (RNA induced silencing complex, RISC).The homologous region of RISC by the mRNA that transcribes with allogenic gene carries out specificity and combines, and induces the homologous gene degraded in the combining site cutting.
The characteristic of the efficient single-minded ground of RNAi inhibition of gene expression makes it obtain using widely aspect gene functional research.The more important thing is that the RNAi technology not only demonstrates irreplaceable superiority in functional genome research, and become possibility for some disease that is difficult to treat provides a kind of new treatment means.Virus disease, especially the disease that is caused by HIV, HBV and HCV retroviral infection is having a strong impact on people's health, and present treatment means can't be effected a radical cure.The RNAi The Application of Technology may provide the means of better treatment and prevention.Hepatitis B virus belongs to hepadnavirus, its reproduction process is template with cccDNA, under the effect of host RNA polymerase II, is transcribed into the mRNA of several different length, wherein the mRNA of 3.5kb contains whole genetic information on the HBV dna sequence dna, is called pregenome RNA.The latter enters cytoplasm of liver as template, under the effect of HBV reversed transcriptive enzyme, and synthetic minus-strand dna; Be template again with the minus-strand dna, under the effect of HBV archaeal dna polymerase, synthetic positive chain DNA, the partially double stranded cyclic DNA of formation filial generation is assembled into complete HBV at last, is released into outside the liver cell.The partially double stranded cyclic DNA of filial generation in the kytoplasm also can enter in the liver cell nuclear, forms cccDNA again and continues to duplicate.Though as seen HBV is a dna virus, its replicanism and RNA viruses are similar.Utilize siRNA, can be directly at the mRNA of HBV, make it that specificity degraded take place, thus blocking-up from the mRNA of HBV to the DNA minus strand the reverse transcription process and various must proteinic translation process, reach the effect of anti-HBV.Existing marketed drug Interferon, rabbit and nucleoside analog more are expected to thoroughly remove hepatitis B virus more near the root of hepatitis B virus.
At present domestic and international several research groups have reported and have successfully used the RNA perturbation technique has suppressed hepatitis B virus efficiently in external and experimentation on animals infection duplication.Inhibiting rate to hepatitis B surface antigen content in the mice serum can be up to more than 90%.Viral DNA in the mouse liver and rna transcription also can effectively be suppressed.These data show that the RNA perturbation technique is the novel method of very promising treating hepatitis B, and the delivery system of multi-form and composition is adopted in these experiments mostly.Be published among the Mechanisms and strategies for effective delivery ofantisense and siRNA oligonucleotides on the Nucleic Acids Research magazines of 2008 36 12 phases of volume and reported the multiple RNA of being applicable to delivery system, but because these delivery system transfection efficiencies are low, cost is higher and the existence of problem such as cytotoxicity has limited it in Clinical Application.Seeking a kind of siRNA delivery system more economical, more high transfection efficiency and better security is RNAi urgent problem in clinical application.
Summary of the invention
Primary and foremost purpose of the present invention provides the shRNA of a specific specificity at hepatitis B virus, to solve the anti-HBV medicine of present clinical application, and the dissatisfactory problem of other RNAi class medication effect especially.Described shRNA has following sequence:
5′-GCAGUUUACUAGUGCCAUUUGUUUCAAGAGAACAAAUGGCACUAGUAAACUGUU-3′
The dna sequence dna that is used to transcribe this shRNA is:
Positive-sense strand:
5′-CACCGCAGTTTACTAGTGCCATTTGTTTCAAGAGAACAAATGGCACTAGTAAACTGTTTTTTG-3′；
Antisense strand:
5′-GATCCAAAAAACAGTTTACTAGTGCCATTTGTTCTCTTGAAACAAATGGCACTAGTAAACTGC-3′
The target sequence of this shRNA identification is at China and Asia HBV oligogene type B, C genotype, has also considered the operability that (D type) estimated in cell experiment, has selected the common ground in B, C, three kinds of hypotype sequences of D.
Second purpose of the present invention provides the recombinant adeno-associated virus gene therapy vector that is used to carry aforementioned shRNA, to improve transfection efficiency, allows to transcribe out the more effective and people's gene group integration of DNA energy after transfection of shRNA.This recombined glandulae correlation viral vectors has following feature:
1) carrier of described recombinant adeno-associated virus carries promotor and the terminator that can transcribe out shRNA in vivo.
2) carrier of described recombinant adeno-associated virus comprises various serotypes, particularly 1 type, 2 types, 8 types and mosaic type.
The 3rd purpose of the present invention provides the preparation method of the recombined glandulae correlation viral vectors that carries aforementioned shRNA, comprises the steps:
1) syntheticly suppresses the dna sequence dna of active siRNA conversion by having HBV.
2) the synthetic dna clone is gone into obtain the recombinant adeno-associated virus skeleton plasmid in the adeno-associated virus skeleton plasmid that can transcribe out siRNA in vivo, described adeno-associated virus skeleton plasmid includes but not limited to pAAV-MCS.
3) the helper plasmid cotransfection of recombinant adeno-associated virus skeleton plasmid and adeno-associated virus is gone into to pack out in the packing cell of adeno-associated virus and obtain recombinant adeno-associated virus, the helper plasmid of described adeno-associated virus includes but not limited to pAAV-RC and pHelper, and described adeno-associated virus packing cell includes but not limited to human embryo kidney (HEK) 293T cell.
Last purpose of the present invention provides aforementioned shRNA and aforementioned recombined glandulae correlation viral vectors thereof and treats and/or prevents purposes in the disease of viral infection medicine in preparation.
Preferred implementation as recombined glandulae correlation viral vectors of the present invention the invention has the advantages that:
1) selected shRNA at HBV B, C, D genotype S gene, with shRNA by behind the recombinant adeno-associated virus transfection HepG2.2.15 cell, not only can effectively suppress the expression of HBsAg and HBeAg in the cell conditioned medium, also can effectively suppress the expression with HBV mRNA of duplicating of HBV DNA in the cell, and compare with lamivudine with the marketed drug Interferon, rabbit of present treatment hepatitis B, restraining effect is more obvious, and restraining effect is stable, and the time length is longer.
2) selected the delivery vector of gland relevant viral vector as shRNA, the characteristics of gland relevant viral vector maximum are: not pathogenic, fool proof.After carrying the gland relevant viral vector transfection HepG2.2.15 cell of shRNA, transfection efficiency is higher, and cytotoxicity is very little simultaneously.
In the present invention: described shRNA can be used for preparing the medicine or the preparation of the relevant any disease of prevention or treatment hepatitis B and hepatitis B virus infection by the RNAi at the HBV gene of recombined glandulae correlation viral vectors mediation.
Description of drawings
Fig. 1, interference plasmid pGPU6-GFP-shRNA structural representation.
Can transcribe out the order-checking collection of illustrative plates of the DNA of shRNA among Fig. 2, the interference plasmid pGPU6-GFP-shRNA.
Fig. 3, recombinant adeno-associated virus skeleton plasmid structural representation.
Fig. 4, recombinant adeno-associated virus transfection efficiency comparison diagram: a are the recombinant adeno-associated virus infected group, and b is empty infection group and viral infection group
Fig. 5, different treatment are to the effect of hepatitis B virus S antigen presentation.Be illustrated as different pharmaceutical and carry the siRNA recombinant adeno-associated virus that suppresses HBV and act on behind the HepG2.2.15 cell different time influence HBsAg level in its cell conditioned medium.No. 1 is control group, is that phalangeal cell deals with PBS under condition of equivalent; Be the Interferon, rabbit processing No. 2; Be the lamivudine processing No. 3; Be recombinant adeno-associated virus transfection processing No. 4.Ordinate zou represents that the antigen levels of different treatment group accounts for the percentage of control group.Mean value ± the SD of three repeated experiments of each data representation among the figure.
Fig. 6, different treatment are to the effect of hepatitis B virus e antigen presentation.Be illustrated as different pharmaceutical and carry the siRNA recombinant adeno-associated virus that suppresses HBV and act on behind the HepG2.2.15 cell different time influence HBeAg level in its cell conditioned medium.No. 1 is control group, is that phalangeal cell deals with PBS under condition of equivalent; Be the Interferon, rabbit processing No. 2; Be the lamivudine processing No. 3; Be recombinant adeno-associated virus transfection processing No. 4.Ordinate zou represents that the antigen levels of different treatment group accounts for the percentage of control group.Mean value ± the SD of three repeated experiments of each data representation among the figure.
The effect of hepatitis B virus DNA level in Fig. 7, the different treatment pair cell.Be illustrated as different pharmaceutical and carry the siRNA recombinant adeno-associated virus that suppresses HBV and act on the influence of hepatitis B virus DNA level in the cell after 8 days of HepG2.2.15 cell.No. 1 is control group, is that phalangeal cell deals with PBS under condition of equivalent; No. 2 is empty virus; Be recombinant adeno-associated virus transfection processing No. 3.Ordinate zou is represented the hepatitis B virus DNA copy number of different treatment group.Mean value ± the SD of three repeated experiments of each data representation among the figure.
The effect of hepatitis B virus mRNA level in Fig. 8, the different treatment pair cell.Be illustrated as different pharmaceutical and carry the siRNA recombinant adeno-associated virus that suppresses HBV and act on the influence of hepatitis B virus mRNA level in the cell after 8 days of HepG2.2.15 cell.No. 1 is control group, is that phalangeal cell deals with PBS under condition of equivalent; No. 2 is empty virus; Be recombinant adeno-associated virus transfection processing No. 3.Ordinate zou represents that the hepatitis B virus mRNA level of different treatment group accounts for the percentage of control group.Mean value ± the SD of three repeated experiments of each data representation among the figure.
Fig. 9, the toxic influence of different treatment pair cell.Be illustrated as different pharmaceutical and carry the siRNA recombinant adeno-associated virus that suppresses HBV and act on behind the HepG2.2.15 cell different time its Cytotoxic influence.No. 1 is control group, is that phalangeal cell deals with PBS under condition of equivalent; Be the Interferon, rabbit processing No. 2; Be the lamivudine processing No. 3; Be recombinant adeno-associated virus transfection processing No. 4.Ordinate zou is represented the light absorption value of cell.Mean value ± the SD of three repeated experiments of each data representation among the figure.
Figure 10, different treatment compared the hepatitis B virus S antigen presentation restraining effect time length.Be illustrated as different pharmaceutical and carry the siRNA recombinant adeno-associated virus that suppresses HBV and act on behind the HepG2.2.15 cell different time influence HBsAg in its cell conditioned medium and HBeAg level.No. 1 is control group, is that phalangeal cell deals with PBS under condition of equivalent; Be the Interferon, rabbit processing No. 2; Be the lamivudine processing No. 3; Be recombinant adeno-associated virus transfection processing No. 4.Ordinate zou represents that the antigen levels of different treatment group accounts for the percentage of control group.Mean value ± the SD of three repeated experiments of each data representation among the figure.
Figure 11, different treatment compared the hepatitis B virus e antigen presentation restraining effect time length.Be illustrated as different pharmaceutical and carry the siRNA recombinant adeno-associated virus that suppresses HBV and act on behind the HepG2.2.15 cell different time influence HBsAg in its cell conditioned medium and HBeAg level.No. 1 is control group, is that phalangeal cell deals with PBS under condition of equivalent; Be the Interferon, rabbit processing No. 2; Be the lamivudine processing No. 3; Be recombinant adeno-associated virus transfection processing No. 4.Ordinate zou represents that the antigen levels of different treatment group accounts for the percentage of control group.Mean value ± the SD of three repeated experiments of each data representation among the figure.
Embodiment
Embodiment 1: the synthetic and Sequence Identification that can transcribe out the DNA of shRNA.
According to the common ground in HBV genotype B, C, three kinds of hypotype sequences of D, by the respective target sequence of bioinformatics method selection HBV, design can be transcribed out the dna sequence dna of shRNA in vivo at target sequence, and the sequence of DNA is as follows:
Positive-sense strand:
5′-CACCGCAGTTTACTAGTGCCATTTGTTTCAAGAGAACAAATGGCACTAGTAAACTGTTTT
TTG-3′
Antisense strand:
5′-GATCCAAAAAACAGTTTACTAGTGCCATTTGTTCTCTTGAAACAAATGGCACTAGTAAAC
TGC-3 ', synthetic interference plasmid pGPU6-GFP-shRNA, the synthetic and order-checking identification of dna sequence by Shanghai JiMa pharmacy Technology Co., Ltd is as Fig. 1 and 2.
Embodiment 2: the structure of recombinant adeno-associated virus skeleton plasmid
With the pGPU6-GFP-shRNA carrier is template, 5 '-GAATTCATGGTGAGCAAGGGCGAGGA-3 ' and 5 '-AAGCTTAAGGTCGGGCAGGAAGAGGG-3 ' is the upstream and downstream primer, the GFP-shRNA-U6 fragment of amplification plasmid pGPU6-GFP-shRNA, and the site of introducing restriction enzyme EcoR I and HindIII.Amplification condition is: 94 ℃ of pre-sex change 3min, and 94 ℃ of sex change 30sec, 65 ℃ of annealing 1min, 72 ℃ are extended 2min, totally 30 circulations, last 72 ℃ of reaction 5min.The PCR product reclaims the back and inserts the pGEM-T carrier, after order-checking is identified correctly, go into after pAAV-MCS plasmid (Stratagene company) double digestion and order-checking (worker is given birth in Shanghai) identify positive recombinant clone with EcoR I and HindIII double digestion directed cloning, called after recombinant vectors rAAV-shRNA-GFP is as Fig. 3.
Embodiment 3: the packing of recombinant adeno-associated virus
With 3 * 10
6Individual 293T cell inoculation is in the culture dish of 10cm in diameter, when degree to be converged reaches 70-80%, use rAAV-shRNA-GFP, pAAV-RC (Stratagene company) and pHelper (Stratagene company) the three plasmid co-transfection 293T cells of the method for liposome and calcium phosphate respectively with each 5-20ug, simultaneously with the empty virus of the negative contrast packing of pAAV-MCS plasmid pAAV, renew bright substratum behind the 6h, during the metamorphosis and the substratum change in color of observation of cell.After continue cultivating 66-72h again, with the cell shovel with cell from culture dish, scrape the back together with media transfer to the centrifuge tube of 15ml.Cell suspension is carried out dry ice-ethanol bath and 37 ℃ of water-baths of four each 10min back and forth, and vortex is even after each freeze thawing.10000 leave heart 10min under the room temperature, collect supernatant and get virus stock solution used with the 0.22um membrane filtration ,-80 ℃ of preservations.Whether virus, detecting with the PCR method simultaneously has wild virus to duplicate (RCL) if being carried out thermal source detection, microorganism detection, guaranteeing does not have thermal source, microbial contaminations such as no bacterium, fungi, virus, and no wild virus duplicates.
Embodiment 4: the detection of recombinant adeno-associated virus transfection efficiency
Get recombinant virus and empty each 200 μ l direct infection 293T cell of virus, behind the cultivation 48h, the fluorescence inverted microscope is observed the expression of green fluorescent protein GFP, and the transfection efficiency of recombinant virus is up to 90%, as Fig. 4 a.And empty infection group and viral infection group is not observed obvious green fluorescence, as Fig. 4 b.
Embodiment 5: the detection of recombinant adeno-associated virus vitro inhibition efficient
With Hep G2.2.15 cell is model, with every hole 500 μ l, 5 * 10
4Individual Hep G2.2.15 cell inoculation 24 orifice plates, after treating cell attachment, IFN-α and medicine final concentration with 1 * 106copies/ cell adding reorganization AAV virus, 10KU/ml is the 100ug/ml lamivudine respectively, and the blank group is set simultaneously, and each laboratory sample is provided with 3 multiple holes.Get the cell culture fluid supernatant 2-8 days every days respectively after the transfection, euzymelinked immunosorbent assay (ELISA) detects the secretory volume of HBsAg and HBeAg, extracted the DNA and the mRNA of cell on the 8th day, fluorescence quantitative PCR method detects DNA and mRNA expression amount, the result shows that reorganization AAV virus all has good inhibitory effect to HBV secreted two kinds of antigen HBsAg and HBeAg, and it is the most obvious to suppress effect in the 6th day, more consistent with time-histories aspect result to two kinds of antigenic restraining effect degree simultaneously, the inhibition effect of Interferon, rabbit and lamivudine is medium.The average copy number of recombinant virus infection group HBV DNA is from 14.41 * 10
6Copies/ml drops to 3.30 * 10
6Copies/ml, inhibiting rate reach (77.10 ± 0.84) %, the inhibiting rate of HBV mRNA are reached (96.85 ± 1.49) %, as Fig. 5,6,7 and 8.
Embodiment 6: the Cytotoxic detection of recombinant adeno-associated virus
With Hep G2.2.15 cell is model, with every hole 50ul, 1 * 10
4Individual Hep G2.2.15 cell is layered in 96 orifice plates, treat cell attachment after, respectively with 1 * 10
6IFN-α and medicine final concentration that the copies/ cell adds reorganization AAV virus, empty virus, 10KU/ml are the 100ug/ml lamivudine, and the blank group is set simultaneously.Use CCK-8 test kit () to detect the cytotoxicity of each sample every day behind the application of sample, detects 7 days altogether.Concrete steps are the CCK-8 of each sample well adding 10ul, after continuing to cultivate 30min, survey light absorption value at the 450nm place.The growth curve basically identical of reorganization AAV virus, empty virus and blank illustrates that its pair cell does not have toxicity substantially, as Fig. 9.
Embodiment 7: the detection of recombinant adeno-associated virus vitro inhibition action time
With Hep G2.2.15 cell is model, with every hole 500 μ l, 5 * 10
4Individual Hep G2.2.15 cell inoculation 24 orifice plates, treat cell attachment after, respectively with 1 * 10
6IFN-α and medicine final concentration that the opies/ cell adds reorganization AAV virus, 10KU/ml are the 100ug/ml lamivudine, and the blank group is set simultaneously, and each laboratory sample is provided with 3 multiple holes.10th, got the cell culture fluid supernatant in 20 and 30 days respectively, euzymelinked immunosorbent assay (ELISA) detects the secretory volume of HBsAg and HBeAg, the result shows that Interferon, rabbit and lamivudine do not suppress effect substantially when 10 days left and right sides, and getting restraining effect, recombinant adeno-associated virus in the time of the 30th day, still can keep certain level, as Figure 10 and 11.
DNA?SEQUENCE?LISTING
＜110〉Beijing Bio-Tech Development Co., Ltd. of Beijing Sanyuan Gene Engineering Co. Ltd.
＜120〉at the shRNA of hepatitis B virus and carry its recombinant adeno-associated virus
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RNA?SEQUENCE?LISTING
＜110〉Beijing Bio-Tech Development Co., Ltd. of Beijing Sanyuan Gene Engineering Co. Ltd.
＜120〉at the shRNA of hepatitis B virus and carry its recombinant adeno-associated virus
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Claims (6)
1. shRNA at hepatitis B virus, its RNA sequence is:
5′-GCAGUUUACUAGUGCCAUUUGUUUCAAGAGAACAAAUGGCACUAGUAAACUGUU-3′
2. the purposes of the described shRNA of claim 1 in the medicine of the relevant disease of preparation prevention or treatment hepatitis B and hepatitis B virus infection.
3. dna sequence dna that is used to transcribe the described shRNA of claim 1, its dna sequence dna is: positive-sense strand:
5′-CACCGCAGTTTACTAGTGCCATTTGTTTCAAGAGAACAAATGGCACTAGTAAACTGTTTTTTG-3′
Antisense strand:
5′-GATCCAAAAAACAGTTTACTAGTGCCATTTGTTCTCTTGAAACAAATGGCACTAGTAAACTGC-3′
4. a recombinant adeno-associated virus gene therapy vector is characterized in that, described recombined glandulae correlation viral vectors carries described dna sequence dna of claim 3 and suitable promotor and terminator.
5. the purposes of the described recombinant adeno-associated virus gene therapy vector of claim 4 in the pharmaceutical preparation of the relevant disease of preparation prevention or treatment hepatitis B and hepatitis B virus infection.
6. the preparation method of the described recombined glandulae correlation viral vectors of claim 4 comprises the steps:
(a) syntheticly suppress the dna sequence dna of active shRNA conversion by having HBV,
(b) the synthetic dna clone is gone into obtain the recombinant adeno-associated virus skeleton plasmid in the adeno-associated virus skeleton plasmid that can transcribe out shRNA in vivo,
(c) the helper plasmid cotransfection of recombinant adeno-associated virus skeleton plasmid and adeno-associated virus is gone into to pack out in the packing cell of adeno-associated virus and obtain recombined glandulae correlation viral vectors.
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| CN104603272B (en) * | 2012-07-06 | 2017-11-07 | 宝生物工程株式会社 | The cell of adeno-associated virus vector can be produced |
| CN107446923A (en) * | 2017-08-13 | 2017-12-08 | 中国人民解放军疾病预防控制所 | RAAV8 CRISPR SaCas9 systems and the application in treating hepatitis B medicine is prepared |
| CN107446923B (en) * | 2017-08-13 | 2019-12-31 | 中国人民解放军疾病预防控制所 | rAAV8-CRISPR-SaCas9 system and application thereof in preparation of hepatitis B treatment drug |
| CN113186224A (en) * | 2021-04-29 | 2021-07-30 | 中国人民解放军陆军军医大学士官学校 | MicroRNA-27a with hepatitis B virus replication inhibition activity and application thereof |
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