CN100560725C - A kind of method of utilizing pSilencer plasmid quickly constructing siRNA carrier - Google Patents
A kind of method of utilizing pSilencer plasmid quickly constructing siRNA carrier Download PDFInfo
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- CN100560725C CN100560725C CNB2007100153331A CN200710015333A CN100560725C CN 100560725 C CN100560725 C CN 100560725C CN B2007100153331 A CNB2007100153331 A CN B2007100153331A CN 200710015333 A CN200710015333 A CN 200710015333A CN 100560725 C CN100560725 C CN 100560725C
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Abstract
The invention discloses a kind of method of the pSilencer of utilization plasmid quickly constructing siRNA carrier.The structure that comprises subcloning vector is used for the synthetic method of the siRNA of rapid screening process, to the steps such as rapid screening method of the positive colony that contains siRNA.Carrier construction method with respect to routine; this method can be more effective; quicker; more economical acquisition is needed less than disturbing genophore; be particularly useful for gene chip; SSH, extensive gene primary dcreening operation result's such as SAGE functional verification analysis, and the research of seeking the RNA interference mechanism that effectively suppresses sequence siRNA etc.
Description
Technical field
The present invention relates to a kind of method of quickly constructing siRNA carrier, relate in particular to a kind of method of in the pSilencer carrier system, utilizing pSilencer plasmid quickly constructing siRNA carrier.
Technical background
The RNA perturbation technique is a kind of powerful tool of research gene function, and " Science " magazine is called RNAi " important breakthroughs in 2002 " (Couzin, 2002).Therefore Andrew Fire and Craig Mello find especially and obtain Nobel Prize in medicine in 2006.The technology of preparing of siRNA mainly comprises: chemical synthesis, the structure of siRNA expression vector, the in-vitro transcription method, preparation siRNA mixture, siRNA expresses box, wherein the siRNA that generates of vector construction method can be in cell several weeks of continuous expression even longer time, and accepted by the molecular biology experiment personnel easily.The product pSilencer of Ambion company series plasmid is the common carrier of carrying siRNA.It contains the rna plymerase iii promotor, ampicillin resistance gene, colibacillary replication origin.After needing reticent gene fragment to insert rna plymerase iii promotor site, change in the mammalian cell, can efficiently express hair fastener shape RNA, thereby suppress the expression of goal gene, in the research that utilizes little perturbation technique inhibition goal gene, be widely used, especially be fit to macrocyclic research.But in carrying the new carrier process of goal gene, structure usually exposes the shortcoming of himself:
1, owing in selecting siRNA target spot process, be difficult to once reach the inhibition effect of the best, therefore, in the application process of reality, often select several target spots to suppress, thus must synthetic several siRNA fragments.If it is very uneconomic that siRNA of each structure removes the ready-made linearization plasmid kit of the company of buying.
2, the existing conventional construction process is, by HindIII and BamH1 double digestion pSilencer cut, and utilizes ligase enzyme that siRNA fragment and linearization carrier are coupled together again.And in implementation process, have a lot of details to have to consider.In fact,, be difficult to, in follow-up connection procedure, cause the recirculation rate to increase, bring great difficulty for the screening of positive colony the thorough double digestion of plasmid because the enzyme of two kinds of restriction enzymes is cut the difference of efficient; In addition, because former pSilencer plasmid itself carries the dna fragmentation that is about 60-70bp, thereby increased the difficulty of positive colony screening, cut or the method for PCR all is difficult to distinguish the DNA band of 10bp difference of only having an appointment at common Agarose glue even SDS-PAGE gel electrophoresis with enzyme, and extremely uneconomic especially by the method screening positive clone of order-checking.
3, the conventional way of the synthetic of siRNA gene fragment is to synthesize the upstream and downstream primer of about 70bp respectively, by annealing, produces the restriction enzyme site of HindIII and BamHI then, is connected in the pSilencer plasmid again.Because the synthetic fragment is longer, and is higher by aforesaid method synthetic siRNA cost.
Given this, set up a kind of can be fast and effectively, timesaving and can reducing greatly makes up siRNA carrier purpose cost, that reach vector construction, novel siRNA carrier construction method is very necessary.
Summary of the invention
At the deficiencies in the prior art, the problem to be solved in the present invention provides a kind of method of the pSilencer of utilization plasmid quickly constructing siRNA carrier.This kind siRNA carrier construction method can fast and effeciently reach the purpose of vector construction, and can reduce the cost that makes up the siRNA carrier greatly.
The method of utilizing pSilencer plasmid quickly constructing siRNA carrier of the present invention, form by following steps:
(1) the synthetic and polishing reaction of siRNA gene fragment:
At the indoor design complementary sequence of siRNA gene fragment, add restriction enzyme site and the protectiveness base of HindIII and BamHI at the siRNA two ends, with the PCR enzymic disposable polishing short dna fragment of high-fidelity; And avoid containing in the siRNA gene target sequence restriction enzyme site of HindIII and BamHI;
(2) preparation of subcloning vector:
Select one of following scheme: 1. on former pSilencer carrier, insert long segment, cut the otherness screening positive clone of product by PCR or enzyme; 2. the fragment that contains special restriction enzyme site is cut the otherness screening positive clone of product by enzyme; 3. produce subclone by ligation;
(3) the siRNA gene fragment is connected with the single stage method of subcloning vector:
Is 1 with subcloning vector and synthetic siRNA gene fragment by mole concentration ratio value: 2-10 is positioned in the same endonuclease reaction system, after enzyme is cut end, and 65 ℃ of deactivation restriction enzymes, the salt concn of attenuating T4DNA ligase enzyme damping fluid is carried out ligation;
(4) contain the screening of the positive colony of siRNA carrier:
The method of cutting by PCR or enzyme will insert the segmental siRNA carrier of 70 ± 4bp and subcloning vector distinguishes.
In the above-mentioned method of utilizing pSilencer plasmid quickly constructing siRNA carrier: the base sequence complementation of upstream and downstream gene 3 ' end parts, the upstream gene sequence that relates to as step (1) is:
5’-GCGGATCCGCTGGGCATCAGTGAGGACATTCAAGAGATGTCCTCACTGATGCC-3’
The downstream gene sequence is:
5’-GCAAGCTTTTCCAAAAAACTGGGCATCAGTGAGGACATCTCTTGAATGTCCTC-3’
Wherein: the PCR reaction is progressively lowered the temperature to reach annealing, the purpose of polishing siRNA gene fragment by 95 ℃ to 55 ℃.
In the method for the above-mentioned pSilencer of utilization plasmid quickly constructing siRNA carrier: the selection principle of the described subcloning vector constructing plan of step (2) is: the double enzyme site that contains HindIII and BamHI is beneficial to make up, and can positive colony be screened enough simple electrophoretic techniques in follow-up siRNA vector selection process.
In the method for the above-mentioned pSilencer of utilization plasmid quickly constructing siRNA carrier: the described subcloning vector of step (3) is preferably 1 with synthetic siRNA gene fragment by mole concentration ratio value: 3-7 mixes.
In the above-mentioned method of utilizing pSilencer plasmid quickly constructing siRNA carrier: the described PCR reaction conditions of step (4) preferably: 95 ℃, 5min; 95 ℃, 30s; 58 ℃, 45s, 72 ℃, 45s; 72 ℃, 10min; Totally 35 circulations.
The siRNA carrier that utilizes the method for the invention to make up is identical with the siRNA carrier of the pSilencer series of ordinary method acquisition.But,
1) owing to shortened the synthetic gene fragment, make siRNA gene fragment synthetic expense reduce 66% at least;
2) by the structure of subclone, solved the problem that positive colony siRNA carrier is difficult to screen, filter out the positive colony that carries the siRNA gene by a PCR reaction;
3) save the step of vector construction, thereby reduced the probability of makeing mistakes, saved the structure time.
In a word; invent the carrier construction method of described method with respect to routine; can be more effective; quicker; the needed little interference genophore of more economical acquisition is particularly useful for to gene chip SSH; extensive gene primary dcreening operation result's such as RACE functional verification analysis, and the research of seeking the RNA interference mechanism that effectively suppresses sequence siRNA etc.
Description of drawings
The technological line of the structure of Fig. 1 subcloning vector and siRNA vector construction
At first be built in the pSilencer3.1-H-neo expression vector by the gene fragment of double digestion with the 329bp size, subclone as screening siRNA expression vector, then, PCR enzyme by high-fidelity is with siRNA gene fragment polishing, at last, by a double digestion and a ligation siRNA is building up in the expression vector.
Fig. 2 double digestion produces the 329bp fragment
1, DL2000, DNA marker; 2, double digestion plasmid mU6pro produces the fragment of 329bp.
The sequencer map of Fig. 3 subclone
Subclone order-checking collection of illustrative plates after the irrelevant sequence of insertion 329bp.
Fig. 4 carries the positive colony PCR screening process of siRNA carrier
1-10 is the PCR product of carrier to be measured, therefrom selects the clone that the molecular weight size is about 300bp and checks order as positive colony.
The sequencer map of Fig. 5 pSilencer-bc1212 expression vector
PSilencer-bc1212 expression vector sequencing result after the siRNA sequence of insertion bc1212 is consistent with synthetic siRNA sequence.
Embodiment
1, the synthetic and PCR polishing reaction of siRNA gene fragment:
1) according to the sequence of bc1212 gene find its interference base because of action target spot (GCTGGGCATCAGTGAGGAC), according to the specification sheets of pSilencer plasmid construction synthetic on, the downstream gene sequence.Be included in sequence in the middle of add ring-like base sequence, add the restriction enzyme site of HindIII and BamHI in upstream and downstream, and add the protectiveness base so that follow-up enzyme is cut (synthetic by Shanghai Bo Ya company) at the restriction enzyme site two ends.
Upstream gene sequence (53bp):
5’-GCGGATCCGCTGGGCATCAGTGAGGACATTCAAGAGATGTCCTCACTGATGCC-3’
Downstream gene sequence (53bp):
5’-GCAAGCTTTTCCAAAAAACTGGGCATCAGTGAGGACATCTCTTGAATGTCCTC-3’
2) PCR reaction polishing siRNA gene fragment:
Pyrobest DNA Polymerase (purchasing company): 2.5U/0.5ul in Takara
Upstream gene sequence: 5ug
Downstream gene sequence: 5ug
10×Pyrobest?Buffer：10ul
10mM?dNTP：3ul
Replenish deionized water to final volume 100ul
The PCR program: 95 ℃, 5min; 93 ℃, 1min; 91 ℃, 1min; 89 ℃, 1min ... 57 ℃, 1min; 55 ℃ of 10min; Get 5ul siRNA gene fragment, electrophoresis is identified in 2% Agarose glue.
2, the preparation of subclone:
1) amplification and the extraction of pSilencer3.1-H-neo and mU6pro carrier;
1, gets the 150ml nutrient solution and pour in the centrifuge tube, centrifugal 30 seconds of 4 ℃ of following 12000g;
2, abandon supernatant, be inverted and liquid is flow to end;
3, bacterial sediment is resuspended to 200 μ l solution I (50mmol/L glucose; 25mmol/LTris-HCL; 10mmol/L EDTA) (needs thermal agitation) in, placed 5-10 minute under the room temperature;
4, solution II (the 0.2mol/L NaOH that adds new preparation; 1%SDS) 400 μ l cover the tight mouth of pipe, and gentleness is put upside down the eppendorf pipe for several times fast, with the mixing content, and ice bath 5 minutes;
5, solution III (the 5mol/L sodium-acetate that adds 300 μ l precoolings; Glacial acetic acid), cover the tight mouth of pipe, and be inverted centrifuge tube, gentle vibration 10 seconds makes the precipitation mixing, in the ice bath 5-10 minute, 4 ℃ the centrifugal 5-10 of following 12000g minute;
6, supernatant liquor moves in the clean eppendorf pipe, adds isopyknic phenol/chloroform (1: 1), vibration mixing, centrifugal 5 minutes of 4 ℃ of following 12000g;
7, water is moved in the clean eppendorf pipe, add isopyknic 5M LiCL
2, centrifugal 10 minutes of 4 ℃ of following 12000g;
8, get supernatant, add the dehydrated alcohol of 2 times of volumes, the vibration mixing was placed in-20 ℃ of refrigerators 20 minutes, and 4 ℃ of following 12000g centrifugal 10 minutes then;
9, abandon supernatant, add 1ml 70% ethanol and wash precipitation once, centrifugal 5 minutes of 4 ℃ of following 12000g;
10, absorb supernatant liquor, vacuum-drying 10 minutes;
11, precipitation is dissolved in the 20 μ l TE damping fluids (pH8.0 contains 20 μ g/ml RNaseA).
2) double digestion of HindIII and BamHI produces the gene fragment of linearization pSilencer-H-neo carrier and 329bp;
BamHI?10ul
HindIII?10ul
10×K?Buffer?30ul
Each 10ug of pSilecer3.1-H-neo/mU6pro
Replenish deionized water to 300ul
37 ℃ of water-baths, enzyme is cut 4h
3) recovery of the gene fragment of linearization pSilencer-H-neo carrier and 329bp and quantitative analysis;
The experimental implementation strictness is carried out with reference to the protocol of QIAquick Gel Extraction Kit (50) (Cat.No.28740 Lot No.12188435);
4) structure of subclone pSilencer329 and evaluation.
Linearization pSilencer-H-neo carrier 50ng
The gene fragment 100ng of 329bp
PEG4000 2.5ul
10×ligase?Buffer 2.5ul
T4DNA ligase enzyme (MBI) 1ul
Replenish deionized water to 25ul, 22 ℃ of connections transform DH5 α competence bacteria, bed board after 1 hour.Cultivated 16 hours screening positive clone, and the evaluation of checking order in 37 ℃ of incubators.
3, being connected of siRNA fragment and subclone
SiRNA gene fragment 200ng
PSilencer329 carrier 50ng
BamHI 10ul
HindIII 10ul
10×K?Buffer 5ul
Replenish deionized water to 50ul, behind 37 ℃ of water-bath effect 12h, 65 ℃, 30min; The centrifugal 10min of 12000rpm gets supernatant 20ul, adds Ligase Bufferl 5ul, PEG4000 2.5ul, T4DNA ligase enzyme 1ul; 22 ℃, 1h.
4, bacterium colony PCR identifies and screening positive clone---the screening of siRNA carrier
To connect product and change among the competence bacteria DH5a, heat-shocked jolts 120min, on shop and the LBA flat board, places 37 ℃ of incubator 16h.With the mono-clonal aseptic inoculation of growing on the bacterium plate in 50ulLB solution, boil 5min after, the centrifugal 10min of 8000rpm gets supernatant as dna solution to be measured.
Dna solution to be measured (unknown concentration) 1ul
M13F(-40)(10nM)：1ul
3.0rev(10nM)：1ul
10mM?dNTP：3ul
Replenish deionized water to 50ul
95℃，5min；95℃，30s；58℃，45s，72℃，45s；72℃，10min；
Totally 35 circulations, concentration are 1% Agarose gel electrophoresis evaluation.
Select the bacterial classification at positive colony place, the evaluation of checking order, the siRNA sequence of inserting on the carrier conforms to fully with former composition sequence comparison.
Claims (3)
1. method of utilizing pSilencer plasmid quickly constructing siRNA carrier, form by following steps:
(1) the synthetic and polishing reaction of siRNA gene fragment:
At the indoor design complementary sequence of siRNA gene fragment, add restriction enzyme site and the protectiveness base of HindIII and BamHI at the siRNA two ends, with the PCR enzymic disposable polishing short dna fragment of high-fidelity; And avoid containing in the siRNA gene target sequence restriction enzyme site of HindIII and BamHI;
(2) preparation of subcloning vector:
Select one of following scheme: 1. on former pSilencer carrier, insert suitable fragment, cut the otherness screening positive clone of product by PCR or enzyme; 2. the fragment that contains special restriction enzyme site is cut the otherness screening positive clone of product by enzyme.
(3) the siRNA gene fragment is connected with the single stage method of subcloning vector:
Is 1 with subcloning vector and synthetic siRNA gene fragment by mole concentration ratio value: 2-10 is positioned in the same endonuclease reaction system, after enzyme is cut end, and 65 ℃ of deactivation restriction enzymes, the salt concn of attenuating T4DNA ligase enzyme damping fluid is carried out ligation;
(4) contain the screening of the positive colony of siRNA carrier:
The method of cutting by PCR or enzyme will insert the segmental siRNA carrier of 70 ± 4bp and subcloning vector distinguishes;
Wherein: the base sequence complementation of upstream and downstream gene 3 ' end parts in the above-mentioned method of utilizing pSilencer plasmid quickly constructing siRNA carrier, the upstream gene sequence that relates to as step (1) is:
5’-GCGGATCCGCTGGGCATCAGTGAGGACATTCAAGAGATGTCCTCACTGATGCC-3’
The downstream gene sequence is:
5’-GCAAGCTTTTCCAAAAAACTGGGCATCAGTGAGGACATCTCTTGAATGTCCTC-3’。
3. utilize the method for pSilencer plasmid quickly constructing siRNA carrier according to claim 1, it is characterized in that: the described subcloning vector of step (3) is 1 with synthetic siRNA gene fragment by mole concentration ratio value: 3-7 mixes.
4. utilize the method for pSilencer plasmid quickly constructing siRNA carrier according to claim 1, it is characterized in that: the described PCR reaction conditions of step (4) is: 95 ℃, and 5min; 95 ℃, 30s; 58 ℃, 45s, 72 ℃, 45s; 72 ℃, 10min; Totally 35 circulations.
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| CN1552876A (en) * | 2003-12-18 | 2004-12-08 | 济南市中心医院 | Targetted liver cancer AFP gene siRNAs expression carrier and its constructing method and use |
| CN1869237A (en) * | 2006-05-26 | 2006-11-29 | 武汉大学 | Construction method of bismall molecule interference RNA carrier and its application |
| CN1974759A (en) * | 2006-07-26 | 2007-06-06 | 吉林大学 | Attenuated salmonella transporting recombinant plasmid and its application in treating tumor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1552876A (en) * | 2003-12-18 | 2004-12-08 | 济南市中心医院 | Targetted liver cancer AFP gene siRNAs expression carrier and its constructing method and use |
| CN1869237A (en) * | 2006-05-26 | 2006-11-29 | 武汉大学 | Construction method of bismall molecule interference RNA carrier and its application |
| CN1974759A (en) * | 2006-07-26 | 2007-06-06 | 吉林大学 | Attenuated salmonella transporting recombinant plasmid and its application in treating tumor |
Non-Patent Citations (4)
| Title |
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| RNA干扰技术的原理与应用. 马鹏鹏等.中国组织化学与细胞化学杂志，第12卷第2期. 2003 |
| RNA干扰技术的原理与应用. 马鹏鹏等.中国组织化学与细胞化学杂志，第12卷第2期. 2003 * |
| 两种高效RNA干涉载体系统的构建及应用. 杨明等.生物化学与生物物理进展，第32卷第4期. 2005 |
| 两种高效RNA干涉载体系统的构建及应用. 杨明等.生物化学与生物物理进展，第32卷第4期. 2005 * |
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