CN101210253B - Optimized small fragment RNA vector system and construction method thereof - Google Patents
Optimized small fragment RNA vector system and construction method thereof Download PDFInfo
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Abstract
The invention provides a modification method for an optimized short-hairpin RNA (shRNA) vector and a preparation method thereof. The method comprises the following steps of: (1) constructing an optimized shRNA vector pGenesil-1.2 by promoter sequence modification, bioelement layout modification and insertion of screened gene with a plasmid vector pEGFP6-1 (Genesil Company) coding multiple shRNAs as skeleton; and (2) chemically synthesizing a DNA template for coding shRNAs and inserting into the pGenesil-1.2, screening clones with double screening method, and sequencing. The invention effectively overcomes the problems of the conventional shRNA vector preparation method in the current scientific research and market, including mutation of promoter 3'-end, difficult identification during construction, and long and high cost of primers for synthesizing template DNA. By the optimized shRNA vector, the promoter for transcribing shRNA has higher transcription efficiency.
Description
Technical field
The utility model relates to biotechnology, and particularly RNA disturbs, and relates in particular to carrier system and construction process thereof that a kind of RNA disturbs optimization (optimization refers to each item transformation for realizing that beneficial effect carried out) the type small fragment RNA (100 bp are with interior RNA) of usefulness.
Background technology
It only is " transition " between DNA and the protein that RNA once had been considered to, but more and more evidences clearly shows, RNA in the process of life role more than we imagine in the early time even more important.
RNA disturbs the discovery of (RNA interference) to make people to the function of RNA regulate gene expression brand-new understanding arranged.The RNA perturbation technique came to light in 1998, and successfully be used for mammalian cell May calendar year 2001, had begun the widespread use of RNA perturbation technique.The RNA perturbation technique is to utilize double-stranded small fragment RNA that goal gene is sealed, and reaches the effect of disturbing goal gene.It is the most ancient genetic immunization monitoring mechanism of life that RNA disturbs.Life keeps health, to resist viral intrusion, antitumorgienesis and keep inheritance stability or the like all possibly be to rely on RNA to disturb to realize, so, the application of research of RNA interferential and RNA perturbation technique is received unprecedented attracting attention.RNA perturbation technique successful Application then (calendar year 2001) just come second that is cited as the world's ten big technical progresses after the nanotechnology.Be cited as first of ten big technical progresses in 2002 again.
The RNA technology is just in develop rapidly; Be the double-stranded RNA of the chemosynthesis of employing May calendar year 2001 when mammalian cell is used RNAi for the first time; Released biology after 1 year and transcribed the synthetic double-stranded RNA, increasing people brings into use the double-stranded RNA that plasmid vector is expressed after 2003.But plasmid vector exists many technological deficiencies and difficult point at present.The accurate insertion at promotor 3 ' end transcription initiation site for the template DNA of realizing the purpose small fragment RNA in the constructing plan of the small fragment RNA expression vector of current main-stream is that the part base of promotor 3 ' is suddenlyd change to introduce a restriction enzyme site mostly.In addition; Because this template DNA is merely about 60 bases usually; This cuts for the enzyme of recombinant vectors and identifies and to have brought difficulty, as under the situation of cutting evaluation without enzyme, directly carrying out determined dna sequence, too high non-recombination fraction then can cause experimental period long with the expending of funds.In order to overcome this problem; In some newer scheme, adopt in the synthetic dna profiling the extra enzyme of introducing to cut and identified the site; This is providing one preferably in the qualification program; But make the base number of synthetic dna profiling surpass 60, go up thereby cause dna primer synthetic cost and mutation rate to have largely.
Summary of the invention
The object of the invention: make up a kind of efficient stable the coding small fragment RNA carrier and its supporting preparation scheme is provided; The conventional preparation coding promotor 3 ' distal process that small fragment RNA vector was faced becomes in current scientific research field and the market to overcome; Identify difficulty in the building process; Synthetic template DNA primer is long, problems such as cost height, and the structure with a kind of optimization makes the promotor of transcribing small fragment RNA that the higher efficient of transcribing arranged simultaneously.
The invention technical scheme: (1) is that skeleton carries out promoter sequence with many shRNA plasmid vectors of the coding pEGFP6-1 of crystalline substance match company, thereby the coding small fragment RNA vector pGenesil-1.2 of optimization is constructed in a series of transformations such as biological elements layout and screening-gene insertion.(2) the required dna profiling of each small fragment RNA of chemical synthesis coding and being built among the pGenesil-1.2 utilizes the dual screening scheme screening and cloning in this system then and identifies the structure work of accomplishing through order-checking.(3) will make up successful coding small fragment RNA expression vector measures through many-sided helpfulness evaluation and practical application effect.
Beneficial effect: utilize BbsI in (1) the utility model; Esp3I; The digestion site of restriction enzymes such as BsaI on recognition site next door and the digestion site sequence do not have specific characteristic; 3 ' end in the purpose promotor is introduced the required sticky end of dna profiling that inserts the coding small fragment RNA with these restriction endonucleases; And terminal bases accordings to natural promotor 3 ' terminal sequence fully, thereby guarantees that promotor 3 ' end has no sudden change, and this provides assurance for initial stability and the efficient of promoter transcription; (2) utilize BbsI in the utility model; Esp3I; Digestion with restriction enzyme site sequences such as BsaI do not have specific characteristics; Inserting that the both sides, site are designed to use with a kind of restriction endonuclease but to digest the sticky end that different, get final product thereby in the preparation process, only need digest, thereby simplify the vector digestion step with a kind of restriction endonuclease; (3) in template DNA insertion site, introduce the label protein expression cassette in advance in the carrier of the utility model, thereby get rid of the non-recombinant clone of recirculation to a great extent, effectively reduce the later stage and identify workload; (4) introduce a kind of particular design in the insertion site of dna profiling 3 ' end in the carrier of the utility model; Make only after the dna profiling of chemosynthesis successfully inserts carrier to form correct recombinant clone, just can form one, thereby under the situation that does not increase synthetic DNA template base number, provide effective enzyme to cut qualification program in the unexistent site of primary template correspondence position; (5) in the carrier of the utility model each functional element carried out optimizing and arranged, thereby do not increased under the prerequisite of any functional element, made the enhanser of CMV can be used, improved it and transcribe efficient for the promotor of small fragment RNA.
Description of drawings
Figure one is for being template with pEGFP6-1; The synoptic diagram that the CMV-EGFP-SV40 expression cassette is increased out with the method for PCR; Add AseI through upstream primer respectively at this expression cassette 5 ' end, BbsI 2., the EcoRI site; And simultaneously the AseI of CMV promotor 5 ' end is suddenlyd change, add the PstI site through downstream primer at this expression cassette 3 ' end.
Figure two is the synoptic diagram through AseI/PstI double digestion linearizing pEGFP6-1 carrier.
Figure three is for coupling together the synoptic diagram that makes up new recombinant plasmid vector pEGFP6-1-ml with linearizing pEGFP6-1 and with the CMV-EGFP-SV40 expression cassette of AseI/PstI digestion.
Figure four adds EcoRI site through upstream primer at its 5 ' end for from human genome, amplifying the U6 promotor, adds BbsI respectively 1., SpeI, and SalI site through downstream primer at this expression cassette 3 ' end.Use SalI then, the EcoRI site builds up to this promotor on the pEGFP6-1-ml, forms the synoptic diagram of new recombinant plasmid vector pEGFP6-1-U6.
Figure five adds SpeI site through upstream primer at its 5 ' end for from pUC19 simple, amplifying the LacZ alpha expression cassette of LAC promotor guiding, adds the SalI site through downstream primer at this expression cassette 3 ' end.Use SpeI then, the SalI site builds up to this expression cassette on the pEGFP6-1-U6, forms the synoptic diagram of new recombinant plasmid vector pGenesil-1.2.
Figure six is with BbsI digestion pGenesil-1.2 carrier; Because a BbsI site is respectively arranged on LacZ expression cassette both sides; So expression cassette can digest from pGenesil-1.2; Because the cleavage site base of these two BbsI designs is different, so two sticky ends at carrier two ends are different, recirculation can not take place.
Figure seven is built into the pGenesil-1.2-GAPDHsh of coding GAPDH shRNA for the dna primer of the good coding shRNA that will anneal connects among the linearizing pGenesil-1.2 with BbsI sticky end paired end through two.
Figure eight is not for to have the BclI site at carrier pGenesil-1.2 and annealed dna template original position, but two paired sticky ends have just been formed a BclI site to be used for the evaluation of recon after combining.
Figure nine is for cutting 1% agarose gel electrophoresis figure behind the recombinant plasmid with the BclI enzyme: in the pGenesil-1.2 initial carrier, be positioned at EGFP 3 ' and hold a unique BclI restriction enzyme site is arranged; After the annealed dna template is inserted, then can go out a new BclI site at the 3 ' end matching that inserts DNA, the base number between these two BclI sites is about 1.8kb.
Among the figure:
1：DL2000?Marker：2000bp、1000bp、750bp、500bp、250bp、100bp
2：pGenesil-1.2-GAPDHsh-1?BclI
3：PGenesil-1.2-GAPDHsh-2?BclI
4：PGenesil-1.2-GAPDHsh-3?BclI
Figure ten detects the result of GAPDH gene in mRNA horizontal expression situation for RT-PCR.Among the figure:
1: blank transfection contrast
2: the negative control of the non-special small fragment RNA of encoding
3: the pGenesil-1-GAPDHsh of the shRNA of transfection conventional type coded interference GAPDH
4: the pGenesil-1.2-GAPDHsh of the shRNA of transfection optimization type coded interference GAPDH
Figure 11 detects the result of GAPDH gene at the protein level expression with Western hybridization.Among the figure:
1: blank transfection contrast
2: the negative control of the non-special small fragment RNA of encoding
3: the pGenesil-1-GAPDHsh of the shRNA of transfection conventional type coded interference GAPDH
4: the pGenesil-1.2-GAPDHsh of the shRNA of transfection optimization type coded interference GAPDH
Figure 12 contrasts the result that the GAPDH in ten kinds of clone suppresses for the pGenesil-1.2 that uses the All Pure Nature promotor in the pGenesil-1 that uses conventional mutant promotor and the prioritization scheme.
Specific embodiments
Further specify the utility model through concrete embodiment below and how to realize, following instance is not used in the scope of restriction the utility model.
One. the carrier transformation
1. be skeleton with the many shRNA carrier pEGFP6-1 that encodes of crystalline substance match company, introduce MCS at the 5 ' end of CMV, and eliminate the shRNA expression cassette of U6 promotor guiding.
1.1 with pEGFP6-1 is template, PCR gets off with the CMV-EGFP-SV40 expression cassette, and introduces required restriction enzyme site at PCR product two ends and the AseI site mutation of holding CMV5 ' falls through primer.
1.1.1 primer sequence
CES-f：
5‘-CATTAATGATCCTTGTCTTCGTCGACGAATTCTAGTTATTCATAGTAATCAATTACG-3’
CES-r：
5‘-AACTGCAGGTTTGGACAAACCACAACTAG-3’
1.1.2 be the upstream and downstream primer with CES-f, CES-r respectively, be template with plasmid pEGFP6-1, with Pfu Turbo (Stratagene) the CMV-EGFP-SV40 expression cassette fragment (PCR signal see figure one) that increases:
1.2 with PstI/AseI two-step approach digestion pEGFP6-1, reclaimed big fragment (seeing figure two), with the PCR product in the last step of PstI/AseI two-step approach digestion.
1.3 the endonuclease bamhi that will go up in the step connects (seeing figure three), and gets 10ul and spend the night and connect product transformed competence colibacillus DH5 α cell, coats on the LB flat board that contains Kana (final concentration is 30ug/ul) resistance 37 ℃ of thermostat container overnight cultures.
1.4 3 single colony inoculations of picking contain the LB nutrient solution of Kana (final concentration is 30ug/ul) resistance in 5ml from flat board, 37 ℃ of constant temperature shaking table overnight cultures.
1.5 extract plasmid with small-scale plasmid extraction test kit, and identify recombinant plasmid with the EcoRI/PstI double digestion respectively, choose positive colony and carry out dna sequencing, with the right-on called after pEGFP6-1-ml of sequence
2. 5 ' of CMV end inserts the shRNA expression cassette of hU6 promotor guiding in pEGFP6-1-ml.
2.1 from the human gene group DNA,, and introduce required restriction enzyme site at PCR product two ends through primer with pcr amplification hU6 promotor.
2.1.1 primer sequence
hU6-f：5‘-GGAATTCAAGGTCGGGCAGGAAGAGG-3’
hU6-r：5‘-GCGTCGACCGACTAGTGAAGACTCGGTGTTTCGTCCTTTCCAC-3’
2.2.2 be the upstream and downstream primer with hU6-f, hU6-r respectively, with human gene group DNA template, with Pfu Turbo (Stratagene) the hU6 expression cassette fragment that increases.
2.2, reclaimed required fragment with the EcoRI/SalI two-step approach digestion pEGFP6-1-ml and the PCR product in a last step.
2.3 the endonuclease bamhi that will go up in the step connects (seeing figure four), and gets 10ul and spend the night and connect product transformed competence colibacillus DH5 α cell, coats on the LB flat board that contains Kana (final concentration is 30ug/ul) resistance 37 ℃ of thermostat container overnight cultures.
2.4 3 single colony inoculations of picking contain the LB nutrient solution of Kana (final concentration is 30ug/ul) resistance in 5ml from flat board, 37 ℃ of constant temperature shaking table overnight cultures.
2.5 extract plasmid with small-scale plasmid extraction test kit, and identify recombinant plasmid with the EcoRI/SalI double digestion respectively, choose positive colony and carry out dna sequencing, with the right-on called after pEGFP6-1-U6 of sequence.
3. 3 ' of hU6 end inserts the LacZ alpha fragment expression frame of LAC promotor guiding in pEGFP6-1-U6.
3.1 from the pUC19 simple (having removed the MCS of pUC19) of crystalline substance match company with pcr amplification LacZ alpha fragment expression frame, and pass through primer at PCR product two ends the required restriction enzyme site of introducing
3.1.1 primer sequence
LacZ-f：5‘-ACGCGTCGACGTCGGGGCTGGCTTAACTATGCGG-3’
LacZ-r：5‘-GGACTAGTTGCAGCTGGCACGACAGGTTTCCC-3’
3.2.2 be the upstream and downstream primer with LacZ-f, LacZ-r respectively, be template with pUC19 simple, with Pfu Turbo (Stratagene) the hU6 expression cassette fragment that increases.
3.2, reclaimed required fragment with SpeI/SalI two-step approach digestion pEGFP6-1-U6 and the PCR product in the last step.
3.3 the endonuclease bamhi that will go up in the step connects (seeing figure five), and gets 10ul and spend the night and connect product transformed competence colibacillus DH5 α cell, coats on the LB flat board that contains Kana (final concentration is 30ug/ul) resistance 37 ℃ of thermostat container overnight cultures.
3.4 3 single colony inoculations of picking contain the LB nutrient solution of Kana (final concentration is 30ug/ul) resistance in 5ml from flat board, 37 ℃ of constant temperature shaking table overnight cultures.
3.5 extract plasmid with small-scale plasmid extraction test kit, and identify recombinant plasmid with the SpeI/SalI double digestion respectively, choose positive colony and carry out dna sequencing, with the right-on called after pEGFP6-1.2 of sequence.
Two. coding is used for RNA interferential shRNA preparing carriers instance
Describe with the shRNA preparation of expression vectors with interference below to people GAPDH
1. sequences Design.
Target site:
5’-GTATGACAACAGCCTCAAG-3′
Explain: this target site bibliographical information site, resist efficient greater than 60%.
Conventional design of primers:
GAPDH1-1
5’-GATCCGTATGACAACAGCCTCAAGTTCAAGACGCTTGAGGCTGTTGTCATACTTTTTTGAATTCA-3’
GAPDH1-2
5’AGCTTGAATTCAAAAAAGTATGACAACAGCCTCAAGCGTCTTGAACTTGAGGCTGTTGTCATACG-3’
Primer length is 65bp.
Optimize the carrier design of primers:
GAPDH2-1
5’-CACCGTATGACAACAGCCTCAAGTTCAAGACGCTTGAGGCTGTTGTCATACTTTTTT-3’
GAPDH2-2
5’GATCAAAAAAGTATGACAACAGCCTCAAGCGTCTTGAACTTGAGGCTGTTGTCATAC-3’
Primer length is 57bp.
2.DNA templa-primer is synthetic
The synthetic required dna profiling primer of this instance of Ying Jun company in Shanghai.
3.DNA templa-primer annealing: dissolve above-mentioned Synthetic 2 0D strand target gene fragment with 50ul annealing buffer; Respectively get 2ul+16ul annealing buffer mixing; 94 ℃ of water-bath annealing naturally cool to room temperature, respectively get 1ul annealing product+99ul H20 and do 100 times of dilutions.
4. vector digestion (shown in figure six):
Get 1ul pGenesil-1.2 carrier and cut 3 hours with 10U BbsI1 enzyme, 1% sepharose reclaims big fragment.
5. will digest good carrier and be connected (shown in figure seven) with the annealing product; And get 10ul and spend the night and connect product transformed competence colibacillus DH5 α cell; Coat contain Kana (final concentration is 30ug/ul) resistance and scribble X-Gal and the LB flat board of IPTG on, 37 ℃ of thermostat container overnight cultures.
6. the screening of blue hickie: when the carrier recirculation then its bacterium colony be blue, and after the annealing fragment was inserted pGenesil-1.2, the LacZ alpha expression cassette on the carrier will be replaced, bacterium colony is white.
7. 3 single colony inoculations of white of picking contain the LB nutrient solution of Kana (final concentration is 30ug/ul) resistance in 5ml from flat board, 37 ℃ of constant temperature shaking table overnight cultures.
8. extract plasmid with small-scale plasmid extraction test kit, and identify recombinant plasmid with BclI respectively.
In the pGenesil-1.2 initial carrier, be positioned at EGFP 3 ' end a unique BclI restriction enzyme site is arranged;, the annealed dna template then can risk a new BclI site (shown in figure eight) by the interface behind PolyA after inserting; Base number between these two BclI sites is about 1.8kb
Enzyme is cut the result and is seen accompanying drawing nine:
Enzyme is cut the result and is shown that endonuclease bamhi is consistent with the expection size, and three clones are positive colony.
9. choose positive colony and carry out dna sequencing, the right-on called after pEGFP6-1.2-GAPDHsh of sequence.
Three. the coding small fragment RNA vector helpfulness of optimization is estimated and practical application effect is measured
1. the synthetic primer cost reduces mensuration.
The synthetic cost of dna primer be in the cost of current structure coding small fragment RNA vector except that human cost the highest cost of accounting weight, account for more than 70% of total cost usually.Be limited to dna primer synthetic technical limitation at present, the cost of synthetic long segment primer (60bp-80bp) is high than small segment primer (8bp-59bp).Be that benchmark compares the synthetic cost of the dna primer that makes up the small fragment RNA plasmid vector of encoding before and after optimizing with the synthetic synthetic price of primer of discussing Shanghai Ying Jun biotech company of the dna primer of present main flow below.
Calculation formula is:
Synthetic primer cost=wall scroll primer base number * 2 * every base price
Conventional scheme synthetic primer cost=63 * 2 * 5.5 (synthetic fragment＞60 price)=715 yuan
Prioritization scheme synthetic primer cost=57 * 2 * 2 (synthetic fragment＜60 price)=228 yuan
The cost rate of descent=(715-228)/715=68.1%
The plasmid vector that is a coding of the every preparation of prioritization scheme wall scroll shRNA can be saved 487 yuans, and the synthetic primer cost descends 68.1%
2. be prepared into power and improve mensuration
The prioritization scheme that the utility model provides is embodied in two aspects to the raising that is prepared into power; One provides dual recombinant screen scheme to reduce the ratio of nonrecombinant; The 2nd, synthetic primer length drops to 60 bases with after interior, helps reducing the resultant fault rate.
Figure ten is the statistic data of relevant situation in the brilliant match company actual fabrication.
Figure ten
Data show among the figure, deliver the recon rate of the positive colony of order-checking after the dual screening scheme screening in prioritization scheme and brought up to 100% from 62%, and the accuracy of synthetic primer has brought up to 97% from 73%.
Figure ten is for conventional preparation scheme and optimize the sequencing result comparative analysis of preparation scheme: the clone is delivered the reliable scheme of order-checking for the checking recon, and the possibility of result of order-checking be (1) nonrecombinant, is the clone of carrier recirculation generation; (2) recon, but the synthetic DNA template of inserting is wrong, because RNA disturbs sequence is had very high requirement, can not comprise any sudden change usually, so there is the sudden change of any base to be the clone of failure; (3) the synthetic right-on recon of base.
The recon ratio is that recon accounts for the ratio in all samples of delivering order-checking, and the entirely true ratio of synthetic primer sequence is the ratio that the quantity of the right-on recon of composition sequence accounts for the recon sum.
3. the pGenesil-1.2-GAPDHsh that constructs in the step 2 is applied to detect to the effects of jamming of GAPDH gene in the HEK293 cell.
3.1 with pGenesil-1.2-GAPDHsh carrier and each control vector the said to specifications transfection HEK293 of METAFECTENEPRO cell with German biontex company; Cultivate after 48 hours with flow cytometer through on the carrier with the green fluorescent protein label sub-elect the cell that is transfected into carrier, the cell that sub-elects is used for subsequent experimental.
3.2 measure GAPDH expression of gene situation in the mRNA level with RT-PCR.
3.2.1 the description of product of pressing Invitrogen company extracts total RNA of sample with Trizol.
3.2.2 carry out reverse transcription reaction
Reaction system:
Reaction conditions: 42 ℃ of 45min, 95 ℃ of 5min
3.2.3 carry out the PCR reaction to goal gene and internal control gene
Primer sequence:
GAPDH?Forward：
5‘-TCCCATCACCATCTTCCA-3’
GAPDH?Reverse：
5‘-CATCACGCCACAGTTTCC-3’
Beta-actin?Forward：
5‘-TCCTCCCTGGAGAAGAGCTA-3’
Beta-actin?Reverse：
5‘-ATCTCCTTCTGCATCCTGTC-3’
Reaction system:
Reaction conditions:
95℃ 5min
72℃ 7min
4℃ …
3.2.41%Agarose gel electrophoresis
PCR result sees accompanying drawing 11
The result shows that pGenesil-1.2-GAPDH has obvious resistance effect and resists effect than the pGenesil-1-GAPDH of traditional scheme GAPDH in the mRNA level and is significantly increased among the figure.
3.3Western hybridization is measured GAPDH expression of gene situation in protein level mensuration level.
3.3.1 extraction total protein of cell
The centrifugal 10min of cell 2000rpm with collecting discards most of supernatant and dispels resuspended mixing.Respectively get 20 μ l samples, add 10 μ l, 3 * loading Buffer, mixing, boiling water bath 10min, the centrifugal 5min of 15000rpm.
3.3.2 preparation SDS-PAGE running gel
3.3.3 carry out vertical slab gel electrophoresis:
With microsyringe the adding of protein sample equal-volume is gone up in the appearance hole, connect power supply and carry out electrophoresis.Spacer gel 90V, when electrophoresis to bromine indigo plant when spacer gel gets into separation gel, voltage is transferred to 140V.
3.3.4 electrotransfer
3.3.4.1 take off gel from electrophoresis apparatus, remove spacer gel, immersing electricity changeed the damping fluid balance 5 minutes.
3.3.4.2 cut two filter paper and a pvdf membrane, size is coincide with gel.Filter paper is immersed electricity to be changeed in the liquid, and pvdf membrane immersed 100% methyl alcohol 5 seconds, and rinsed with deionized water is once put into electricity again and changeed the damping fluid balance 5 minutes.
3.3.4.3 the installation orifice plate, and relative with the transfer groove positive and negative electrode, put into groove, adding 1*Transfer Buffer electricity changes damping fluid, energized, the 100mA constant current was changeed film 1.5 hours.
3.3.5 hybridization
3.3.5.1 the pvdf membrane that takes a turn for the better is immersed confining liquid (1*TBST+5% skim-milk), and 4 ℃ are spent the night;
3.3.5.2 1*TBST washes film 5min * 4 time;
Resist 3.3.5.3 add one, the room temperature shaking table is hatched 2h;
3.3.5.4 1*TBST washes film 5min * 4 time;
Hatch 1h 3.3.5.5 add two anti-room temperature shaking tables;
3.3.5.6 1*TBST washes film 5min * 4 time;
3.3.6 exposure
3.3.6.1 place the SuperSignal chemical illuminating reagent to react 2min pvdf membrane;
3.3.6.2 make the exposure of X-ray sheet in the darkroom, the ordinary method developing fixing.
The result sees accompanying drawing 12
The result shows that pGenesil-1.2-GAPDH has obvious resistance effect and resists effect than the pGenesil-1-GAPDH of traditional scheme GAPDH at protein level and is significantly increased among the figure.
3.4 the Detection of Stability of prioritization scheme in various kinds of cell system
Though a spot of sudden change to promotor 3 ' can not cause that in most cells significant starting efficiency changes, still there is potential impact in its starting efficiency for promotor, and this influence is significant in specific cells.And the promotor that is used in this prioritization scheme guide shRNA to transcribe accordings to natural series fully, has no sudden change, has then thoroughly eradicated this influence.Below through to using mutant and natural promoter that the inhibition effect comparison of GAPDH is detected the stability of prioritization scheme in various kinds of cell system in ten kinds of clone respectively.
Detected result is seen figure 13
The result representes that the natural promoter in this prioritization scheme keeps identical or slightly high resistance rate than the mutant promotor in most cells system among the figure, in small amounts of cells, has significantly than the resistance rate of mutant and increases.
Claims (8)
1. the small fragment RNA vector system of an optimization; It is characterized in that; With many shRNA plasmid vectors of the coding pEGFP6-1 of crystalline substance match company is that skeleton carries out promoter sequence, biological elements layout and screening-gene and inserts to transform and construct the coding small fragment RNA vector pGenesil-1.2 of optimization; The dna profiling that each small fragment RNA of chemical synthesis coding is required also is built among the pGenesil-1.2; Utilize dual screening scheme screening and cloning and warp order-checking in this system to identify the work of completion structure, utilize BbsI, Esp3I; The digestion site of BsaI restriction enzyme on recognition site next door and the digestion site sequence do not have specific characteristic, guaranteed that promotor 3 ' end has no sudden change when inserting the segmental restriction enzyme site of template DNA introducing.
2. the small fragment RNA vector system of a kind of optimization according to claim 1 is characterized in that inserting the label gene expression frame in advance in the position of the insertion template DNA of carrier, so that a kind of screening mechanism to be provided in the bacterium colony level.
3. the small fragment RNA vector system of a kind of optimization according to claim 1 and 2; Just can form the unexistent restriction enzyme site in original position in the carrier when it is characterized in that providing a kind of design to make having only the template DNA fragment to insert, thereby a kind of effective recon identification of means is provided.
4. the small fragment RNA vector system of a kind of optimization according to claim 1 is characterized in that, on the arrangement of elements of carrier cmv enhancer and hU6 promotor next-door neighbour is arranged, thereby effectively utilizes the enhanser of CMV to improve the starting efficiency of hU6 promotor.
5. the construction process of the small fragment RNA vector system of an optimization is characterized in that: with many shRNA plasmid vectors of the coding pEGFP6-1 of crystalline substance match company is that skeleton carries out promoter sequence, biological elements layout and screening-gene and inserts to transform and construct the coding small fragment RNA vector pGenesil-1.2 of optimization; The dna profiling that each small fragment RNA of chemical synthesis coding is required also is built among the pGenesil-1.2, utilizes dual screening scheme screening and cloning and warp order-checking in this system to identify the structure work of accomplishing then; Utilize BbsI, Esp3I, the digestion site of BsaI restriction enzyme on recognition site next door and the digestion site sequence do not have specific characteristic, guaranteed that promotor 3 ' end has no sudden change when inserting the segmental restriction enzyme site of template DNA introducing.
6. the construction process of the small fragment RNA vector system of a kind of optimization according to claim 5 is characterized in that inserting the label gene expression frame in advance in the position of the insertion template DNA of carrier, so that a kind of screening mechanism to be provided in the bacterium colony level.
7. the construction process of the small fragment RNA vector system of a kind of optimization according to claim 5; Just can form the unexistent restriction enzyme site in original position in the carrier when it is characterized in that providing a kind of design to make having only the template DNA fragment to insert, thereby a kind of effective recon identification of means is provided.
8. the construction process of the small fragment RNA vector system of a kind of optimization according to claim 5; It is characterized in that; On the arrangement of elements of carrier cmv enhancer and hU6 promotor next-door neighbour is arranged, thereby effectively utilize the enhanser of CMV to improve the starting efficiency of hU6 promotor.
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| 刘明社等.干扰Fas受体表达的串联shRNA表达载体的构建.《世界华人消化杂志》.2006,第14卷(第22期),2174-2179. * |
| 李凡东.质粒介导shRNA对心肌细胞kir2.1蛋白表达盒搏动频率的影响.《中国老年学杂志》.2006,第26卷240-242. * |
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