CN101684478A - Method for constructing tandem expression small interfering RNA recombinant lentiviral vector - Google Patents
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Abstract
The invention discloses a method for constructing a tandem expression small interfering RNA recombinant lentiviral vector. The method comprises the following steps of: (1) constructing the siRNA recombinant lentiviral vector with single target spot: a, chemically synthesizing an oligonucleotide chain; b, annealing a sense chain and an anti-sense chain; and c, connecting an annealing fragment to the vector; (2) constructing the siRNA recombinant lentiviral vector with double target spots: a, amplifying a U6/H1-siRNA expression frame from the siRNA recombinant lentiviral vector with single target spot by utilizing the PCR; b, connecting the expression frame to the siRNA recombinant lentiviral vector with single target spot undergoing enzyme digestion by the XhoI; and c, performing the enzymedigestion, identification and screening of the clone forwardly inserted in the expression frame; and (3) constructing the siRNA recombinant lentiviral vector with multiple target points in tandem connection: inserting the U6/H1-siRNA expression frame in the siRNA recombinant lentiviral vector with double target spots to construct the siRNA recombinant lentiviral vector with triple target spots, and obtaining the siRNA recombinant lentiviral vector with multiple target points in tandem connection by repeating the step. The construction method is suitably used for the expression of the siRNA with multiple target genes as well as the research on the multiple target genes which are silent for a long time.
Description
Technical field
The present invention relates to biology field.Relate more specifically to a kind of construction process of tandem expression small interfering RNA recombinant lentiviral vector.
Background technology
(RNA interference RNAi) is meant sequence-specific PTGS (post-transcriptional gene silencing, process PTGS) that is triggered by double-stranded RNA in the RNA interference.At present, RNA interferential mechanism is illustrated substantially: double-stranded RNA endogenous or external source is cut into the siRNA (siRNA) of giving prominence to 21～23nt of 3 ' end with 2 bases by Dicer in tenuigenin, the latter and RNA inductive silencing complex (RNA-induced silencing complex, RISC) combination.SiRNA is unwind in RISC, and wherein antisense strand guides RISC target complementary mRNA with it, and RISC is cutting mRNA in the middle of complementary sequence then, thereby suppresses target gene expression.
Although find so far only 11 years from the RNAi phenomenon, RNAi has been widely used in Basic of Biology research and Application Areas as a kind of gene silencing instrument.5 kinds of methods that prepare siRNA comparatively commonly used at present comprise: 1) chemosynthesis; 2) in-vitro transcription; 3) the disconnected dsRNAs of lengthy motion picture is through RNase III enzyme liberating; 4) the siRNA expression cassette of PCR preparation is expressed in cell; 5) the siRNA expression vector is expressed siRNA in cell.Above method all has relative merits separately: the siRNAs of preceding 4 kinds of methods preparation or siRNA expression cassette are gone into cell through transfection or electricity transduction, can realize the transient gene silence, and be then not too suitable for the albumen that the transformation period is long.The siRNA expression vector then can utilize the antibiotic marker on the carrier to set up metastable long-term gene silencing cell strain, continues to suppress target gene expression.Most siRNA expression vectors utilizes III type rna polymerase promoter to handle siRNA and expresses in mammalian cell, and the III type promotor that adopts usually comprises people source and mouse source U6 promotor and people source H1 promotor.At present, a lot of biotech firms have developed the siRNA expression vector based on U6 or H1 promotor, and representative product comprises: the pSilencer series of Ambion company; The siLentGene series of Promega company; The BLOCK-iT series of Invitrogen company, etc.
The siRNA expression vector can be divided into plasmid vector and virus vector two classes.There is the low shortcoming of transfection efficiency for some difficult cells transfected plasmid vector, so beginning to research and develop virus vector, many in recent years biotech firms express siRNA, it is advantageous that directly the high-level efficiency cells infected carries out the research of gene silencing, avoid owing to the low inconvenience that brings of plasmid transfection efficient.Virus vector commonly used at present comprises: retroviral vector, adenovirus carrier, lentiviral vectors.Wherein lentiviral vectors have all if can infect division and non-division stage cell, can be integrated in advantage such as host genome and quilt application more and more widely.Representative siRNA lentiviral vectors such as pLentiLox 3.7, this carrier contain one and handle siRNA by mouse source U6 promotor and express.
Yet, that has developed at present only is suitable for the expression of the siRNA of single target spot based on the siRNA slow virus expression vector of III type promotor, and increasing fundamental research and applied research need reticent a plurality of target genes simultaneously, in order to address this problem, the present invention has designed the construction process of the lentiviral vectors of a kind of tandem expression siRNA, is applicable to the experimental study and the applied research of a plurality of target genes of long-term silence.
Summary of the invention
The objective of the invention is to be to provide a kind of construction process of tandem expression siRNA recombined lentivirus vector, overcome the limitation that existing siRNA expression vector only is applicable to the siRNA that expresses single target spot, can express siRNA simultaneously at a plurality of target genes, and be suitable for the foundation of stable cell lines, thereby be applicable to the experimental study and the applied research of a plurality of target genes of long-term silence.
The construction process of this tandem expression siRNA recombined lentivirus vector may further comprise the steps:
1, the promoter engineering of lentiviral vectors:
The lentiviral vectors that the applicant transforms is pLentiLox 3.7 (Rubinson and Dillon, NatureGenetics, 2003).This carrier contains the expression that a mouse source U6 promotor is used for controlling siRNA.Promotor for fear of series connection siRNA is single and cause that homologous recombination causes losing of siRNA expression cassette, the applicant replaces the mouse source U6 promotor of this carrier respectively and is people source U6 promotor and people source H1 promotor, and improved lentiviral vectors is called after pLLU6, pLLH1 (Escherichia coli Stbl3/pLLU6CCTCCM 209135 respectively; Escherichia coli Stbl3/pLLH1 CCTCC M 209136, preservation date: on June 25th, 2009, depositary institution: Chinese typical culture collection center, address: China. Wuhan. Wuhan University)
2, the construction process of tandem expression siRNA recombined lentivirus vector
2.1 the structure of the siRNA recombined lentivirus vector of single target spot
2.1.1 the chemical synthetic oligonucleotide chain, form is as follows:
Sense strand: 5 '-(19N)-(TTCAAGAGA)-(N91)-TTTTTTC-3 '
Antisense strand:, and add to go up TCGA to produce Xho1 viscosity protruding terminus at 5 ' end with the sense strand complementation
Annotate: 19N represents 19 base sequences of siRNA target spot, and N19 represents the reverse complementary sequence of 19N;
2.1.2 sense strand, antisense strand annealing form the terminal fragment of band Xho1 sticky end peace;
2.1.3 above fragment is connected to carrier pLLU6 or pLLH1 (carrier is by KspA1 and Xho1 double digestion);
2.1.4 transformed into escherichia coli Stbl3 competent cell;
2.1.5 screening positive clone, and order-checking is identified;
2.2 the structure of the siRNA recombined lentivirus vector of two target spots
2.2.1 utilize PCR " U6/H1-siRNA " expression cassette that from the siRNA recombined lentivirus vector of single target spot, increases, and make it have the Xho1 sticky end with Sal1, Xho1 double digestion
For " U6-siRNA " expression cassette, the PCR primer sequence is as follows:
Upstream primer: U6-siR-1 (5 '-TAACGC
GTCGACCCCCAGTGGAAAGACGCGCA-3 ')
Sal1
Downstream primer: U6-siR-2 (5 '-ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
For " H1-siRNA " expression cassette, the PCR primer sequence is as follows:
Upstream primer: H1-siR-1 (5 '-TAACGC
GTCGACAATTCATATTTGCATGTCGC-3 ')
Sal1
Downstream primer: H1-siR-2 (5 ' ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
2.2.2 " U6/H1-siRNA " expression cassette is connected to the siRNA recombined lentivirus vector of single target spot of cutting through the XhoI enzyme, and transformed into escherichia coli Stbl3 competent cell, bacterium colony PCR screening positive clone;
2.2.3 enzyme is cut the clone of evaluation and screening " insertion of fragment forward "
Because there is the possibility of positive and negative both direction in the segmental insertion of " U6/H1-siRNA " expression cassette, in order to be fit to the structure and the Screening and Identification of series connection siRNA recombined lentivirus vector, this step is only selected the clone that forward inserts.Concrete authentication method is: with Xba1, Xho1 double digestion clone to be measured, establish and do not insert segmental carrier for contrast, if the endonuclease bamhi size greater than contrast, then is " forward insertion "; If the endonuclease bamhi size is identical with contrast, then be " oppositely inserting ";
2.2.4 order-checking is identified
2.3 the structure of the series connection siRNA recombined lentivirus vector of many target spots
" U6/H1-siRNA " expression cassette is inserted into the siRNA recombined lentivirus vector of two target spots, promptly constructs the siRNA recombined lentivirus vector of three target spots,, can obtain the placed in-line siRNA recombined lentivirus vector of many target spots with this construction strategy.
In the application example of one group of interference virus of AIDS, the applicant has made up the series connection siRNA recombined lentivirus vector at 3 target spots, experimental result confirms this lentiviral vectors energy works better, and placed in-line siRNA expression cassette is (referring to the embodiment 3) that works alone.
The invention has the beneficial effects as follows: the lentiviral vectors of (1) tandem expression siRNA provided by the present invention is applicable to reticent a plurality of target genes simultaneously, be suitable for having the fundamental research and the applied research of demand like this, such as: in the treating AIDS field, different target spots with siRNA while a plurality of virogenes of target or the same virogene of target can effectively prevent or delay viral escape; (2) the present invention is suitable for the foundation of stable cell lines, thereby can reticent steadily in the long term target gene; (3) be easy to realize the directed insertion of siRNA expression cassette; (4) it is selective that the lentiviral vectors of tandem expression siRNA provided by the invention provides two kinds of promotors of U6, H1, can avoid to a certain extent causing losing of siRNA expression cassette because of the homologous recombination that repeatedly repeats to cause with a kind of promotor.
Description of drawings
Fig. 1 is the promoter engineering synoptic diagram of lentiviral vectors.Use from the people source U6 of pSilencer plasmid and people source H1 promotor to replace mouse source U6 promotor on the pLentiLox 3.7 respectively, obtain pLLU6, pLLH1 carrier.
Fig. 2 is the structure schema of tandem expression small interfering RNA recombinant lentiviral vector.At first, the oligonucleotide fragment of the coding siRNA of chemosynthesis is connected to pLLU6, pLLH1 carrier, obtains the siRNA recombined lentivirus vector of single target spot by KspA1 and Xho1 restriction enzyme site; Then, pcr amplification " U6/H1-siRNA " expression cassette is connected to the siRNA recombined lentivirus vector of single target spot that the Xho1 enzyme cuts, thereby obtains the siRNA recombined lentivirus vector of two target spots; At last, construction of strategy goes out the recombined lentivirus vector of tandem expression siRNA according to this.
Fig. 3 is the lentiviral vectors application example of tandem expression small interfering RNA.This example is chosen the required host cell membrane PROTEIN C CR5 of hiv gene rev, pol, tat and virus infection cell as target spot, constructs the siRNA recombined lentivirus vector of single target spot, two target spot, three target spots.This sample result shows: the lentiviral vectors energy works better of tandem expression small interfering RNA provided by the invention, and placed in-line siRNA expression cassette works alone.
Embodiment
Below in conjunction with drawings and Examples the present invention is further specified.
Embodiment 1: the promoter engineering of lentiviral vectors
The lentiviral vectors that the applicant transforms is pLentiLox 3.7 (Rubinson and Dillon, NatureGenetics, 2003).This carrier contains the expression that a mouse source U6 promotor is used for controlling siRNA, also contains reporter gene EGFP simultaneously and is convenient to selected by flow cytometry apoptosis.Promotor for fear of series connection siRNA is single and cause that homologous recombination causes losing of siRNA expression cassette, the applicant replaces the mouse source U6 promotor of this carrier respectively and is people source U6 promotor and people source H1 promotor (people source U6 promotor and people source H1 promotor obtain through PCR from product pSilencer2.0_U6 of Ambion company and pSilencer3.0_H1), and improved lentiviral vectors is called after pLLU6, pLLH1 (Escherichia coli Stbl3/pLLU6 CCTCC M209135 respectively; Escherichia coli Stbl3/pLLH1 CCTCC M 209136).The promoter engineering synoptic diagram is seen shown in Figure 1.Concrete transformation process is as follows:
1.1PCR amplification U6 and H1 promoter fragment;
For the U6 promotor, the PCR primer is as follows:
Upstream primer: U6-1 (5 ' ACA
TCTAGACCCCAGTGGAAAGACGCGCAG-3 ')
Xba1
Downstream primer: U6-2 (5 '-ACG
GTTAACGATCCCGCGTCCTTTCCACAA-3 ')
KspA1
For the H1 promotor, the PCR primer is as follows:
Upstream primer: H1-1 (5 ' CGG
TCTAGAAATTCATATTTGCATGTCGCTA-3 ')
Xba1
Downstream primer: H1-2 (5 '-AGC
GTTAACCGAGTGGTCTCATACAGAACTT-3 ')
KspA1
1.2 above promoter fragment is cloned into pEGM-T carrier (available from Promega company) with T-A clone strategy;
1.3, isolate the U6/H1 promoter fragment of band sticky end with Xba1 and the above reorganization of KspA1 double digestion pEGM-T carrier;
1.4, obtain carrier with corresponding sticky end with Xba1 and KspA1 double digestion pLentiLox 3.7;
1.5 the U6/H1 promoter fragment is connected to above carrier, obtain the lentiviral vectors of promoter engineering, respectively called after pLLU6, pLLH1 (Escherichia coli Stbl3/pLLU6 CCTCC M 209135; Escherichia coli Stbl3/pLLH1 CCTCC M 209136).
Embodiment 2: the construction process of tandem expression siRNA recombined lentivirus vector
2.1 the structure of the siRNA recombined lentivirus vector of single target spot (referring to Fig. 2 step I)
2.1.1 the chemical synthetic oligonucleotide chain, form is as follows:
Sense strand: 5 '-(19N)-(TTCAAGAGA)-(N91)-TTTTTTC-3 '
Antisense strand:, and add to go up TCGA to produce Xho1 viscosity protruding terminus at 5 ' end with the sense strand complementation
Annotate: 1) 19N represents 19 base sequences of siRNA target spot, and N19 represents the reverse complementary sequence of 19N;
2) (TCAAGAGA) instruct " ring " zone that generates siRNA precursor shRNA (bobby pin RNA), this sequence is selected from (Brummelkamp et al., Science, 2002)
2.1.2 sense strand, antisense strand annealing form the terminal fragment of band Xho1 sticky end peace;
2.1.3 above fragment is connected to carrier pLLU6 or pLLH1 (carrier is by KspA1 and Xho1 double digestion);
2.1.4 transformed into escherichia coli Stbl3 competent cell (the Stbl3 bacterial strain is available from Invitrogen company);
2.1.5 screening positive clone, and order-checking is identified;
2.2 the structure of the siRNA recombined lentivirus vector of two target spots (referring to Fig. 2 Step II)
2.2.1 utilize PCR " U6/H1-siRNA " expression cassette that from the siRNA recombined lentivirus vector of single target spot, increases, and make it have the Xho1 sticky end with Sal1, Xho1 double digestion
For " U6-siRNA " expression cassette, the PCR primer sequence is as follows:
Upstream primer: U6-siR-1 (5 '-TAACGC
GTCGACCCCCAGTGGAAAGACGCGCA-3 ')
Sal1
Downstream primer: U6-siR-2 (5 ' ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
For " H1-siRNA " expression cassette, the PCR primer sequence is as follows:
Upstream primer: H1-siR-1 (5 '-TAACGC
GTCGACAATTCATATTTGCATGTCGC-3 ')
Sal1
Downstream primer: H1-siR-2 (5 ' ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
2.2.2 " U6/H1-siRNA " expression cassette is connected to the siRNA recombined lentivirus vector of single target spot of cutting through the XhoI enzyme, and transformed into escherichia coli Stbl3 competent cell, bacterium colony PCR screening positive clone;
2.2.3 enzyme is cut the clone of evaluation and screening " insertion of fragment forward "
Because there is the possibility (is forward with closure shown in Fig. 2 Step II) of positive and negative both direction in the segmental insertion of " U6/H1-siRNA " expression cassette, in order to be fit to the structure and the Screening and Identification of series connection siRNA recombined lentivirus vector, this step is only selected the clone that forward inserts.Concrete authentication method is: with Xba1, Xho1 double digestion clone to be measured, establish and do not insert segmental carrier for contrast, if the endonuclease bamhi size greater than contrast, then is " forward insertion "; If the endonuclease bamhi size is identical with contrast, then be " oppositely inserting ";
2.2.4 order-checking is identified
Sequencing primer: 5 '-CAGTGCAGGGGAAAGAATAGTAGAC-3 '
(Rubinson?and?Dillon，Nature?Genetics，2003)；
2.3 the structure of the series connection siRNA recombined lentivirus vector of many target spots (referring to Fig. 2 Step II I)
" U6/H1-siRNA " expression cassette is inserted into the siRNA recombined lentivirus vector of two target spots, promptly constructs the siRNA recombined lentivirus vector of three target spots,, can obtain the placed in-line siRNA recombined lentivirus vector of many target spots with this construction strategy.
Embodiment 3: the lentiviral vectors application example of tandem expression siRNA
Whether can works better for the lentiviral vectors of verifying tandem expression siRNA, the applicant is illustrated with the application example that disturbs virus of AIDS:
In this embodiment, the applicant chooses the required host cell membrane PROTEIN C CR5 of hiv gene rev, pol, tat and virus infection cell as target spot, utilizes the lentiviral vectors of tandem expression siRNA provided by the present invention to construct the siRNA recombined lentivirus vector of single target spot, two target spot, three target spots.SiRNA expression cassette array mode is as shown in the table:
| Single target spot | ??H1?CCR5 ??U6?rev ??U6?pol ??U6?tat |
| Two target spots | ??H1?CCR5_U6?rev ??H1?CCR5_U6?pol ??H1?CCR5_U6?tat |
| Three target spots | ??H1?CCR5_U6?rev_U6?pol ??H1?CCR5_U6?rev_U6?tat ??H1?CCR5_U6?pol_U6?tat |
The applicant with above siRNA recombined lentivirus vector respectively with pNL4-3.Luc.R-E-(Landau NR, Viology, 1995) cotransfection HEK-293T cell, detect the plain enzymic activity of relative fluorescence after 24 hours, the result is (annotate: pNL4-3.Luc.R-E-is the HIV replicon of band luciferase reporter gene, and its levels of replication can be represented by uciferase activity) shown in Fig. 3 (A).The result shows: each siRNA recombined lentivirus vector is to the restraining effect that all has in various degree of duplicating of HIV, wherein the effect of Combination of rev-pol, rev-tat, pol-tat siRNA is higher than its effect of performance separately separately, illustrate that rev, pol, tat siRNA expression cassette can work alone, and action effect has synergistic effect after making up in twos.The concrete outcome analytical data is as shown in the table:
(annotate: CCR5 siRNA expression cassette does not play a role in above-mentioned experiment, because used HEK-293T cell is not expressed the CCR5 membranin, and pNL4-3.Luc.R-E-is that liposome transfection enters cell, need not membrane receptor CCR5 mediation)
In order to verify can working alone of CCR5 siRNA expression cassette, the applicant utilizes constructed siRNA recombined lentivirus vector pLLH1 CCR5 and pLLH1 CCR5_U6 rev_U6 tat to set up stable cell lines (KewalRamani V and Littman DR based on GhostCD4/CCR5, J Virol, 1999) (this clone can stably express membranin CCR5).The CCR5 antibody (available from eBioscience company) of using the PE mark has then carried out streaming detection (result is shown in Fig. 3 (B), and gray area is a sample, the negative contrast of hollow area) to the stable cell lines of above foundation.The result shows: the CCR5 siRNA that is expressed by pLLH1CCR5 and pLLH1 CCR5_U6rev_U6 tat is effective down-regulation of CCR 5 expression level all, and the two no significant difference (reducing 77.5% and 79.9% respectively), thereby explanation: the working alone of the CCR5 siRNA expression cassette in the siRNA recombined lentivirus vector of tandem expression.
In sum, the application example explanation that present embodiment relates to: the lentiviral vectors energy works better of tandem expression siRNA provided by the invention, and placed in-line siRNA expression cassette works alone.
Embodiment 4: the packing of the slow virus of tandem expression siRNA.
4.1 transfection preceding 24 hours shops HEK-293T cell (available from CCTCC) is in the Tissue Culture Dish of a diameter 7.5cm;
4.2 cell degree of converging is advisable with 40～60% during transfection, operates following plasmid co-transfection cell with the calcium phosphate transfection of standard:
4 μ g siRNA recombined lentivirus vectors
2μg?pLP1
2μg?pLP2
2μg?pVSVG
(annotate: pLP1, pLP2, pVSVG are slow virus auxiliary package plasmids, available from Invitrogen company);
4.3 collected viral supernatant (being cell culture medium) respectively in 24 hours, 48 hours, 72 hours after the transfection, and supply substratum; Mix the viral supernatant of collecting for 3 times afterwards;
4.4 virus titer detects: with viral supernatant with 10 times of gradient dilutions, infect the HEK-293T cell respectively and (add polybrene (available from SIGMA company) in the substratum, final concentration is 8 μ g/ml), streaming detects GFP positive cell ratio after 48 hours, and calculates virus titer with the extent of dilution of ratio between 0.1-10%.Embodiment 5: utilize the slow virus of tandem expression siRNA to set up stable cell lines.
5.1 the slow virus with packaging among the embodiment 4 infects Ghost CD4/CCR5 cell with gradient m.o.i., infect streaming detection GFP positive cell ratio after 48 hours, select its ratio 10% following person and carry out aseptic airflow classification (annotate: selection ratio 10% following person why is to be infected again by slow virus and influence the accuracy of subsequent experimental for fear of cell);
The cell of cultivating sorting 5.2 go down to posterity is suitable to cell quantity;
5.3 streaming detects GFP positive cell ratio once more, to determine the purity of stable cell lines.
SEQUENCE?LISTING
＜110〉Wuhan University
＜120〉a kind of construction process of tandem expression small interfering RNA recombinant lentiviral vector
＜130〉a kind of construction process of tandem expression small interfering RNA recombinant lentiviral vector
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Claims (3)
1, a kind of lentiviral vectors pLLU6 of transformation is characterized in that: Escherichia coli Stbl3/pLLU6CCTCC M 209135.
2, a kind of lentiviral vectors pLLH1 of transformation is characterized in that: Escherichia coli Stbl3/pLLH1CCTCC M 209136.
3, a kind of construction process of tandem expression siRNA recombined lentivirus vector may further comprise the steps:
(1) structure of the siRNA recombined lentivirus vector of single target spot:
A, chemical synthetic oligonucleotide chain, form is as follows:
Sense strand: 5 '-(19N)-(TTCAAGAGA)-(N91)-TTTTTTC-3 '
Antisense strand:, go up TCGA to produce Xho1 viscosity protruding terminus in 5 ' end interpolation with the sense strand complementation;
B, sense strand, antisense strand annealing form the terminal fragment of band Xho1 sticky end peace;
C, above fragment is connected to carrier pLLU6 or pLLH1;
D, transformed into escherichia coli Stbl3 competent cell;
E, screening positive clone, and order-checking is identified;
(2) structure of the siRNA recombined lentivirus vector of two target spots:
A, utilize the PCR U6/H1-siRNA expression cassette that from the siRNA recombined lentivirus vector of single target spot, increases, and make it have the Xho1 sticky end with Sal1, Xho1 double digestion
For the U6-siRNA expression cassette, the PCR primer sequence is as follows:
Upstream primer: U6-siR-1 (5 '-TAACGC
GTCGACCCCCAGTGGAAAGACGCGCA-3 ')
Sal1
Downstream primer: U6-sjR-2 (5 '-ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
For the H1-siRNA expression cassette, the PCR primer sequence is as follows:
Upstream primer: H1-siR-1 (5 '-TAACGC
GTCGACAATTCATATTTGCATGTCGC-3 ')
Sal1
Downstream primer: H1-si R-2 (5 ' ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
B, the U6/H1-siRNA expression cassette is connected to the siRNA recombined lentivirus vector of single target spot of cutting through the Xhol enzyme, and transformed into escherichia coli Stbl3 competent cell, bacterium colony PCR screening positive clone;
C, enzyme are cut the clone that evaluation and screening fragment forward inserts, this step is only selected the clone that forward inserts, concrete authentication method is: with Xba1, Xho1 double digestion clone to be measured, be not contrast if insert segmental carrier, the endonuclease bamhi size is greater than contrast, for forward inserts, the endonuclease bamhi size is identical with contrast, is reverse insertion;
D, order-checking are identified;
(3) structure of the series connection siRNA recombined lentivirus vector of many target spots:
The U6/H1-siRNA expression cassette is inserted into the sjRNA recombined lentivirus vector of two target spots, promptly constructs the siRNA recombined lentivirus vector of three target spots,, can obtain the placed in-line siRNA recombined lentivirus vector of many target spots with this construction strategy.
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| WO2012055362A1 (en) * | 2010-10-28 | 2012-05-03 | Benitec Biopharma Limited | Hbv treatment |
| CN103834692A (en) * | 2014-02-27 | 2014-06-04 | 南京医科大学 | Lentiviral vector for expressing lncRNA (long noncoding ribonucleic acid) and application thereof |
| WO2015113511A1 (en) * | 2014-01-29 | 2015-08-06 | 江苏命码生物科技有限公司 | Sirna in tandem expression and uses thereof in treating chronic lymphocytic leukemia |
| CN106434753A (en) * | 2016-07-26 | 2017-02-22 | 沈阳医学院 | TAT-RGD-KDR siRNA fusion gene vector, and construction method and application thereof |
| US9790502B2 (en) | 2010-10-28 | 2017-10-17 | Benitec Biopharma Limited | HBV treatment |
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| AU2003268546A1 (en) * | 2002-09-06 | 2004-03-29 | Massachusetts Institute Of Technology | Lentiviral vectors, related reagents, and methods of use thereof |
| US7763722B2 (en) * | 2003-01-17 | 2010-07-27 | University Of Florida Research Foundation, Inc. | Small interference RNA gene therapy |
| CA2600440A1 (en) * | 2005-03-09 | 2006-09-21 | Baylor College Of Medicine | Direct reversal of the suppressive function of cd4+ regulatory t cells via toll-like receptor 8 signaling |
| CN101113169B (en) * | 2006-07-28 | 2011-07-13 | 中国科学院广州生物医药与健康研究院 | Use of sorting protein SNX10 for suppressing growth of tumour cell |
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2009
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| WO2012055362A1 (en) * | 2010-10-28 | 2012-05-03 | Benitec Biopharma Limited | Hbv treatment |
| CN103370415A (en) * | 2010-10-28 | 2013-10-23 | 本尼特生物制药有限公司 | HBV treatment |
| US9080174B2 (en) | 2010-10-28 | 2015-07-14 | Benitec Biopharma Limited | HBV treatment |
| US9410154B2 (en) | 2010-10-28 | 2016-08-09 | Benitec Biopharma Limited | HBV treatment |
| RU2620966C2 (en) * | 2010-10-28 | 2017-05-30 | Бенитек Биофарма Лимитед | Treatment of hbv infection |
| CN103370415B (en) * | 2010-10-28 | 2017-05-31 | 本尼特生物制药有限公司 | HBV is treated |
| US9790502B2 (en) | 2010-10-28 | 2017-10-17 | Benitec Biopharma Limited | HBV treatment |
| WO2015113511A1 (en) * | 2014-01-29 | 2015-08-06 | 江苏命码生物科技有限公司 | Sirna in tandem expression and uses thereof in treating chronic lymphocytic leukemia |
| US10273481B2 (en) | 2014-01-29 | 2019-04-30 | Jiangsu Mircromedmark Biotech Co., LTD. | SiRNA in tandem expression and uses thereof in treating chronic lymphocytic leukemia |
| CN103834692A (en) * | 2014-02-27 | 2014-06-04 | 南京医科大学 | Lentiviral vector for expressing lncRNA (long noncoding ribonucleic acid) and application thereof |
| CN103834692B (en) * | 2014-02-27 | 2016-03-30 | 南京医科大学 | A kind of lentiviral vectors for expressing lncRNA and application thereof |
| CN106434753A (en) * | 2016-07-26 | 2017-02-22 | 沈阳医学院 | TAT-RGD-KDR siRNA fusion gene vector, and construction method and application thereof |
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