CN103060374B - RNA interference vector based on site-specific recombination, and construction method and application of same - Google Patents
RNA interference vector based on site-specific recombination, and construction method and application of same Download PDFInfo
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Abstract
The invention discloses a RNA interference vector based on site-specific recombination, and construction method and application of the same, belonging to the field of biotechnology. The interference vector provided by the invention sequentially comprises the following connected parts: a homologous recombination arm 1 in homologous recombination with the upstream of a certain intron region of the integrin receptor beta 3 gene of pig, a human U6 promoter sequence, a multiple cloning enzyme digestion site, a human U6 terminator sequence, a new Neomycin resistant gene containing a PCMV (Porcine cytomegalovirus) promoter, a homologous recombination arm 2 in homologous recombination with the downstream of a certain intron region of the integrin receptor beta 3 gene of pig, a replication origin gene, an ampicillin resistant screened gene and a enzyme digestion site for vector linearization. The interference vector provided by the invention realizes accurate targeting to swine-origin cell genome, and facilitates screening, meanwhile, the influence of insertion of segment on the gene expression can be predicted, which is convenient for the following experiment.
Description
Technical field
The present invention relates to a kind of rna interference vector and construction process thereof and application, particularly a kind of rna interference vector and construction process and application based on locus specificity restructuring, belongs to biological technical field.
Background technology
RNA disturbs (RNA interference, RNAi) to refer to the phenomenon of the efficient specificity degraded of high conservative, that brought out by double-stranded RNA (double-stranded RNA, dsRNA), homologous mRNA during evolution.Owing to using the RNAi technology can specific depletion or close the expression of specific gene, so this technology has been widely used in exploring the field of gene of gene function and communicable disease and malignant tumour.
In animal, RNAi can start expression shRNA by U6 to be realized.Current U6 carrier can be expressed in zooblast and be expressed by 2 kinds of modes: transient expression and stably express.Stably express is mainly realized by inserting in genome.Mainly by Gateway technology or carrier radom insertion host cell gene group, complete at present.The defect of these technology is to carry out directly accurately locating of on position, and radom insertion may affect the expression of goal gene.
In order to realize, in stable expression cell line, insert the accurate location of genome position and the expression that does not affect genes involved, the present invention combines with homology target practice technology by U6 promotor, realized the accurate target practice to pig source cell, and provide convenience for screening, can expect the impact on genetic expression after Insert Fragment, for subsequent experimental provides convenience simultaneously.
Summary of the invention
The present invention is that binding site specificity homologous recombination and RNA disturb the specificity that (RNAi) technology is invented to insert the novel RNAi carrier in pig β 3 intron regions.Herein, U6 promotor, in conjunction with the homology technology of practicing shooting, makes genome location more convenient, and can guarantee that the destination gene expression of screened engineering cell is unaffected.
The present invention has adopted following technique means in order to achieve the above object:
A kind of rna interference vector based on locus specificity restructuring of the present invention, it is characterized in that since 5 ' end, comprise the each several part that next coming in order are connected: carry out the homologous recombination arm 1 of homologous recombination with certain upstream, single intron region on integrin receptor β 3 genes of pig, human U_6 promoter sequence, polyclone restriction enzyme site, people U6 terminator sequence, the neomycin resistance gene that contains PCMV promotor, carry out the homologous recombination arm 2 of homologous recombination with certain downstream, single intron region on integrin receptor β 3 genes of pig, replication origin gene, amicillin resistance screening-gene and for the linearizing restriction enzyme site of carrier, the GeneBank accession number of integrin receptor β 3 genes of described pig is NC_010454.3.
Wherein homologous recombination arm 1,2 carries out homologous recombination for certain single intron region on integrin receptor β 3 genes with pig; Human U_6 promoter is used for expressing object shRNA, for stablizing RNAi; PCMV-NEO: for the screening of anti-G418 resistant cell; Replication origin (Origin) provides the Copy Info of carrier in intestinal bacteria; Amp promoter-Amp is for a large amount of propagation of screening positive clone or thalline; At the polyclone restriction enzyme site between human U_6 promoter sequence and people U6 terminator sequence, for enzyme, cut the sticky end exposing between U6 promotor and termination signal, be convenient to insert object ds oligo.
In the present invention, preferred, on integrin receptor β 3 genes described and pig, to carry out the homologous recombination arm 1 of homologous recombination be that extracting from the DNA of pig liver is template in certain upstream, single intron region, by using following primer amplification to obtain:
Primers F: 5 '-AAGTCCGGCAGGTGGAGGATTACC-3 '
Primer R:5 '-CCCAGTGCTGGTTATCACATGGC-3 ';
On integrin receptor β 3 genes described and pig, to carry out the homologous recombination arm 2 of homologous recombination be that extracting from the DNA of pig liver is template in certain downstream, single intron region, by using following primer amplification to obtain:
Primers F: 5 '-GGCCATGTGATAACCAGCACTGGGA-3 '
Primer R:5 '-CCCCCTTGTAGTGGACACAGGAGA-3 '.
In the present invention, preferred, the nucleotide sequence of described carrier is as shown in SEQ ID NO.1.
Further, the present invention also provides a kind of method that builds the rna interference vector described in above any one, it is characterized in that comprising the following steps:
(1) structure of homology arm
Take that to extract be template from the DNA of pig liver, design two pairs of primers homologous recombination arm 1 and homologous recombination arm 2 that homologous recombination used that be used for respectively increasing:
Amplification homologous recombination arm 1 primer used is
F:5’-AAGTCCGGCAGGTGGAGGATTACC-3’
R：5’-CCCAGTGCTGGTTATCACATGGC-3’；
Homologous recombination arm 2 primer used is
F：5’-GGCCATGTGATAACCAGCACTGGGA-3’
R：5’-CCCCCTTGTAGTGGACACAGGAGA-3’。
The fragment that clone obtains is connected respectively on PMD20-T carrier, obtains being connected with respectively recombinant vectors PMD20-T-HA1 and the PMD20-T-HA2 of homologous recombination arm 1 and homologous recombination arm 2;
(2) structure of human U_6 promoter, polyclone restriction enzyme site, people U6 terminator sequence
The carrier of take containing human U_6 promoter, polyclone restriction enzyme site, people U6 terminator sequence is template, pcr amplification obtains the fragment that contains human U_6 promoter, polyclone restriction enzyme site, people U6 terminator, the fragment that clone is obtained is connected on PMD20-T carrier, builds and obtains PMD20-T-U6;
(3) structure of positive screening-gene
Neomycin resistance gene NEO and PCMV promotor are cloned on carrier PGT-V1, and the amplimer of NEO is:
F:5’-ACACGATGATAAGCTTGCCAC-3’
R：5’-TTAGCTAGCGAACCTCTTCGAGGGACCTAATAACTTC-3’；
The amplimer of PCMV promotor is:
F:5’-ACGGGATCCATATACGCGTTGACATTGATTATTGAC-3’
R：5’-GTTCCCGGGAGAGCTCTGCTTATATAGACCTCCC?AC-3’；
(4) structure of skeleton carrier
Synthetic multiple clone site, and be subcloned on pUC57 carrier, building skeleton carrier PUC57-MCS, multiple clone site sequence is as shown in SEQ ID NO.2 or Figure 13;
(5) element assembling
1. after utilizing speI on PMD20-T-HA2 carrier and BamHI site enzyme to cut, homologous recombination arm 2 is connected to skeleton carrier PUC57-MCS upper, is configured to carrier pUC57-2;
2. the assembling of PCMV and NEO gene: by after the PCMV being cloned into and the recovery of NEO gene glue, utilize the XmaI restriction enzyme site on gene that two gene splicings are become to PCMV-NEO, PCMV-NEO is cloned into carrier pUC57-2 above, be configured to carrier pUC57-N2;
3. utilize AgeI and the XmaI on SalI and PMD20-T-HA1 and SalI site on PMD20-T-U6, HA1 is cloned into PMD20-T-U6 above, be built into PMD20-T-HA1-U6; Utilize SalI and ClaI site on PMD20-T-HA1-U6 that object fragment HA1-U6 is cloned into pUC57-N2 above, be configured to object carrier PSN-U6.
In the present invention, preferred, in step (2), build the sequence of the fragment that contains human U_6 promoter, polyclone restriction enzyme site, people U6 terminator obtaining as shown in SEQ ID NO.3.
Further, the present invention also provides the application in building pig source cell RNAi expression vector of rna interference vector described in above any one.
When using vector construction pig source cell RNAi expression vector of the present invention, comprise the following steps:
1, select as required the restriction enzyme site of polyclone position, double digestion carrier, the U6 promotor on exposure PSN-U6 and the sticky end between termination signal.
2, the synthetic required DNA sequence dna of shRNA of transcribing, for example step 1, as when selecting ClaI, SfiI as restriction enzyme site, meets the ds-oligo(DNA of this carrier) as shown in figure 11:
3, the PSN-U6 after synthetic fragment is cut with enzyme is connected, and transform and connect product, picking positive colony sequence verification, sequencing primer is:
5’-G?GACTATCATA?TGCTTACCG-3’。
4, obtain in a large number positive bacteria liquid, large upgrading grain.
5, use the suitable flat end restriction enzyme site carrying on carrier, enzyme is cut carrier, makes it linearizing.
6, electric shock transfectional cell, screening positive clone.
By southern, hybridize checking object fragment and mix genomic copy number, utilize PCR primer to verify whether homology is inserted genomic specificity position.
Accompanying drawing explanation
Fig. 1 is the carrier collection of illustrative plates of recombinant vectors PMD20-T-HA1;
Fig. 2 is the carrier collection of illustrative plates of recombinant vectors PMD20-T-HA2;
Fig. 3 is the carrier collection of illustrative plates of recombinant vectors PMD20-T-U6;
Fig. 4 is the junction fragment of PCMV and NEO;
Fig. 5 is the carrier collection of illustrative plates of recombinant vectors PUC57-MCS;
Fig. 6 is the carrier collection of illustrative plates of recombinant vectors pUC57-2;
Fig. 7 is the carrier collection of illustrative plates of recombinant vectors pUC57-N2;
Fig. 8 is the carrier collection of illustrative plates of recombinant vectors PMD20-T-HA1-U6;
Fig. 9 is the carrier collection of illustrative plates of recombinant vectors PSN-U6;
Figure 10 is for building the outline flowchart of the rna interference vector PSN-U6 with homologous recombination ability;
Figure 11 is the ds-oligo(DNA that meets this carrier) schematic diagram;
Figure 12 is the synthetic required DNA sequence dna of shRNA of transcribing in the embodiment of the present invention;
Figure 13 is synthetic multiple clone site sequence schematic diagram;
Figure 14 is the PCR blob of viscose figure that identifies positive colony;
Figure 15 is southern experimental result;
Figure 16 is engineering cell and the normal mRNA level comparison with reference to α v in cell;
Figure 17 is mRNA amplification curve diagram;
Figure 18 is mRNA solubility curve figure;
Figure 19 is the horizontal statistical graph of mRNA relative expression.
Embodiment
Below by experiment, also the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiment, only for the object of illustration, never limit the scope of the invention.Those of ordinary skills understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, revise, and even equivalence change, but all will fall within the scope of protection of the present invention.
The construction process of 1 one kinds of rna interference vectors of recombinating based on locus specificity of embodiment
1, experiment material
Pig liver tissue: be purchased from Gansu laboratory animal support base
I α v-pENTR/U6 carrier: preserved by this laboratory
PGT-V1: support use by teacher Wang Huayan of Xibei Univ. of Agricultural & Forest Science & Technology
Polyclone restriction enzyme site, uses relevant primer: synthetic by Shanghai biotechnology company limited in experiment
The all ingredients box of using in test is purchased from precious biotechnology (Dalian) company limited, and another XmaI and AgeI are purchased from NEB company.
2, method
The formation of 2.1 carriers
The formation of carrier is as shown in PSN-U6 structure iron spectrogram 9, and its function introduction is as follows:
HA1, HA2: for carrying out homologous recombination with the pig genome β single intron of 3 gene region
HU6(human U_6 promoter): for expressing object shRNA, for stablizing RNAi
PCMV-NEO: for screening anti-G418 resistant cell screening
Origin: the Copy Info of carrier in intestinal bacteria
Amp promoter-amp: for a large amount of propagation of screening positive clone or thalline
NruI, SrfI: before transfection, linearized vector
ClaI, SfiI: for enzyme, cut the sticky end exposing between U6 promotor and termination signal, be convenient to insert object ds oligo.
The clone of 3.1 vector gene original papers
3.1.1 the structure of homology arm
In NCBI, screen satisfactory intron region in pig genome, finally determine a certain specific intron region of integrin receptor β 3 genes (GeneBank accession number is NC_010454.3) that use pig.For two pairs of primers of single intron zone design long-armed and galianconism that homologous recombination used that is used for respectively increasing.
Long-armed (HA1) primer used that increases is
F:5’-AAGTCCGGCAGGTGGAGGATTACC-3’
R：5’-CCCAGTGCTGGTTATCACATGGC-3’；
Amplification galianconism (HA2) primer used is
F：5’-GGCCATGTGATAACCAGCACTGGGA-3’
R：5’-CCCCCTTGTAGTGGACACAGGAGA-3’。
Long-armed and length galianconism is respectively: 7036bp, 2424bp.Long-armed amplification program is: 94 ℃ of 1min, 58 ℃ of 50s, 72 ℃ of 8min(30cycles); 72 ℃ of 8min(over).The amplification program of galianconism is: 94 ℃ of 1min, 59 ℃ of 50s, 72 ℃ of 3min(30cycles); 72 ℃ of 3min(over).The goal gene group source that amplification is used is pig liver tissue.And the fragment that clone is obtained is connected respectively on PMD20-T carrier, carry out sequence verification, finally obtain being connected with respectively long-armed and recombinant vectors PMD20-T-HA1 and PMD20-T-HA2 galianconism, its carrier collection of illustrative plates is as shown in Figure 1 and Figure 2.
3.1.2 the structure of human U_6 promoter, polyclone restriction enzyme site, people U6 terminator sequence
The fragment that the former U6 carrier that contains α v interference fragment building in laboratory of take contains human U_6 promoter, polyclone restriction enzyme site, people U6 terminator sequence as template amplification, the amplimer using is
F:5’-ACCGGTACTGGATCCGGTACCAAGGTC-3’
R：5’-CATCGATGTTTCGTCCTTTCCAC-3’。
Amplification length is 289bp, and amplification program is: 94 ℃ of 1min, 55 ℃ of 50s, 72 ℃ of 3min(30cycles); 72 ℃ of 3min(over).The fragment that clone is obtained is connected on PMD20-T carrier, carry out sequence verification, the fragment sequence that contains human U_6 promoter, polyclone restriction enzyme site, people U6 terminator that clone obtains is as shown in SEQ IDNO.3, wherein contained polyclone restriction enzyme site is ClaI, SfiI, structure obtains PMD20-T-U6, and its carrier collection of illustrative plates as shown in Figure 3.
3.1.3 the structure of positive screening-gene
Positive screening-gene (neomycin resistance gene NEO) and PCMV promotor are cloned on carrier PGT-V1.The amplimer of NEO is: F:5 '-ACACGATGATAAGCTTGCCAC-3 ' R:5 '-TTAGCTAGCGAACCTCTTCGAGGGACCTAATAACTTC-3 '; Its amplification program is 94 ℃ of 1min, 59 ℃ of 50s, 72 ℃ of 1min30s(30cycles); 72 ℃ of 8min(over); Length is 1226bp.The amplimer of PCMV is F:5 '-ACGGGATCCATATACGCGTTGACATTGATTATTGAC-3 ' R:5 '-GTTCCCGGGAGAGCTCTGCTTATATAGACCTCCC AC-3 '; Its amplification program is 94 ℃ of 1min, 57 ℃ of 50s, 72 ℃ of 1min(30cycles); 72 ℃ of 8min(over); Length is 615bp, and the junction fragment of PCMV and NEO as shown in Figure 4.
3.1.4 the structure of skeleton carrier
The gene order of resulting each gene original paper of this experimental basis, design is not present in the multiple clone site of the restriction enzyme site on these genes, multiple clone site is served to Hai Shenggong synthetic, and be subcloned on pUC57 carrier, build skeleton carrier PUC57-MCS, multiple clone site sequence is as shown in SEQ ID NO.2 or Figure 13, and PUC57-MCS carrier collection of illustrative plates as shown in Figure 5.
3.1.5 element assembling
The subclone that carries out gene by step is as shown in Figure 10 assembled into each gene original paper on skeleton carrier PUC57-MCS.
(1) after utilizing speI on PMD20-T-HA2 carrier and BamHI site enzyme to cut, galianconism is connected to skeleton carrier PUC57-MCS upper, is configured to carrier pUC57-2, send sequence verification, carrier collection of illustrative plates as shown in Figure 6.
(2) assembling of PCMV and NEO gene: by after the PCMV being cloned into and the recovery of NEO gene glue, utilize the XmaI restriction enzyme site on gene that two gene splicings are become to PCMV-NEO, while utilizing design primer, the BamHI comprising on PCMV-NEO and NheI and the BglII above pUC57-2 and SpeI, PCMV-NEO is cloned on carrier pUC57-2, be configured to carrier pUC57-N2, send sequence verification, carrier collection of illustrative plates as shown in Figure 7.
(3) utilize AgeI and the XmaI on SalI and PMD20-T-HA1 and the SalI site on PMD20-T-U6, HA1 is cloned into PMD20-T-U6 above, be built into PMD20-T-HA1-U6, send sequence verification, carrier collection of illustrative plates as shown in Figure 8.Utilize SalI and ClaI site on PMD20-T-HA1-U6 that object fragment HA1-U6 is cloned into pUC57-N2 above, be configured to object carrier PSN-U6, and carry out sequence verification, as shown in Figure 9, the sequence of carrier is as shown in SEQ ID NO.1 for carrier collection of illustrative plates.Restriction enzyme and reaction conditions that in each subclone step, each carrier is used are as shown in table 1.
Restriction enzyme and reaction conditions that in each subclone step of table 1, each carrier is used
Note: two restriction endonucleases that connected by "/" represent to carry out double digestion simultaneously; By ", " two restriction endonucleases connecting represent that two enzyme distribution enzymes cut
Enzyme is cut after product recovery, uses T4-DNA ligase enzyme, and 16 degrees Celsius of connections of spending the night, then transform DH5 α, picking positive colony.
The application of embodiment 2 carrier of the present invention in building the reticent expression vector of pig source cell
1, build and be used for expressing shRNA, and by homologous recombination, insert the reticent expression vector of pig source cell of the genomic specific position of pig source cell
(1) use ClaI and SfiI double digestion carrier, the U6 promotor on exposure PSN-U6 and the sticky end between termination signal.Reaction conditions is 1*M damping fluid, adds 2 kinds of enzymes simultaneously, 30 ℃ of 1.5h, then temperature is adjusted to 50 ℃ of 1.5h, reclaims enzyme and cuts product.
(2) synthesize and transcribe the required DNA sequence dna of shRNA, as shown in figure 12:
(3) PSN-U6 after synthetic fragment is cut with enzyme is connected, and transform and connect product, picking positive colony sequence verification, sequencing primer is:
5’-G?GACTATCATA?TGCTTACCG-3’。
(4) obtain a large amount of positive bacteria liquid, large upgrading grain.
(5) use the suitable flat end restriction enzyme site carrying on carrier, enzyme is cut carrier, makes it linearizing.
(6) utilize Eppendorf Multiporator electroporation, select 800V, 150 μ s, 2 times, electric shock transfectional cell mixed solution (800 μ L cell 1*10
8/ mL+30 μ g linearized vector).After electricity turns, add the 20mL perfect medium dilution electricity thing of changing the line of production, then cell culture fluid is spread to six orifice plates (1.5mL/ hole).Electricity turns after 48h, adds G418 and screens (300 μ g/mL), within 6 days, inserts afterwards the unicellular of object fragment and grows up to larger clone, the not necrocytosis of Insert Fragment.Aseptic micrurgy picking mono-clonal, adds in 96 orifice plates and cultivates.
According to cell growth condition, be transferred to successively 48 orifice plates, 24 orifice plates, 12 orifice plates, 6 orifice plates.Collect the cell in 6 orifice plates, extract the genomic dna of 180 monoclonal cells of institute's picking, use the mono-clonal that detects primer screening site-directed integration, screening positive cell mono-clonal primer pair: F primer(is positioned at the Auele Specific Primer of carrier upstream): 5 '-AGCGAAACATCGCATCGAGC-3 '; R primer(is positioned at the Auele Specific Primer of integrating fragment downstream): 5 '-ACCACCATCTACTCAGCTCTGC-3 ', identifies the blob of viscose figure of positive colony as shown in figure 14.The marker using is 250bp DNA ladder marker, and fragment is followed successively by 250bp, 500bp, 750bp, 1000bp, 1500bp, 2250bp, 3000bp, 4500bp.Near marker is the normal contrast with reference to cell.From electrophorogram, can judge have a strain positive colony (expection size is 3215bp) between 3000-4500bp to occur a positive band, control cells and other mono-clonal all do not have this band.Preliminary judgement is needed engineering cell.After this band is cut, glue reclaims, and is connected on PMD-20-T and checks order, and shown in the following SEQ ID of sequence results NO.4, sequencing result, with predicted the outcome identical, is judged to be positive site-directed integration clone.
2, the copy number of southern checking Insert Fragment in gene
Use DIG-High Prime DNA Labeling and Detection Starter Kit II(Roche AppliedScience) PCR is identified to the genomic dna of positive mono-clonal and normal control cell carries out southern detection, determine that it inserts copy number.
1. probe preparation: the NEO gene that while using carrier construction element, clone obtains is as probe sequence (1213nt, sequence is as shown in SEQ ID NO.5).
2. genomic dna preparation and processing: use Genome DNA purification kit (Promega) to extract the genomic dna that PCR identifies positive mono-clonal and normal control cell, use ClaI(to be positioned at the single restriction enzyme site on carrier) be not present in the restriction enzyme site on carrier with BsiWI() use neb bufferIII to carry out 37 ℃ of double digestions that spend the night to extracted genome.
The step of using above-mentioned materials to provide according to test kit specification sheets, carries out southern detection to genomic dna.
What experimental result: Figure 15 showed is southern experimental result, according to band, can find out, PCR identifies that a strip-like developing pipe appears in positive mono-clonal, and normal control cell develops without band.Proof PCR identifies that positive cell inserts genomic copy number for single copy.
3, the checking of carrier interference level of the present invention
3.1. acceptor gene interference effect checking
Use GAPDH as reference gene, the mRNA level of the gene that inserted interference fragment is disturbed is carried out relative quantification, and the primer using is as shown in table 2 below
Table 2
Use Trizol to extract total RNA of engineering cell and normal control cell, use NanoDrop2000 (Thermo) to carry out concentration determination to extracted RNA, use ThermoScript II M-MLV (Takara) to carry out reverse transcription to total RNA, be totally 15 microlitres, each concentration of component is the total RNA of 1.5 μ g, 0.5mM dNTP each, 5 μ M oligo-dT primers, 11 μ l 5 * M-MLV buffer, 300U M-MLV.Use SYBR Premix ExTaq (Takara) test kit, it is quantitative that Mx3005P QPCR system (Stratagene) carries out relative fluorescence to goal gene, and response procedures is: 95 ℃ of 30s; 95 ℃ of 5s, 60 ℃ of 30s(40cycles); 95 ℃ of 15s, 60 ℃ of 60s, 95 ℃ of 15s.Use 2-Δ Δ Ct method to measure the acceptor gene α v relative expression level of engineering cell.Comparison result shows, in engineering cell, the mRNA level of α v is 0.463 of the normal mRNA level with reference to α v in cell, as shown in figure 16, amplification curve, solubility curve and relative expression's level are respectively as shown in Figure 17,18,19 for result.
3.2. cell indirect ELISA is measured the expression level of α v subunit
The cell suspension of control cells and engineering cell is added in 96 orifice plates to (1.0 * 10
5cells/well), cell absorption is outwelled residual media after spending the night, and uses the how anti-4 ℃ of absorption 1h of rabbit of the α v albumen of 50 microlitre 1:500 dilutions, outwell adsorption liquid, use cold PBS to wash cell, 4 ℃ of absorption 1h of mouse-anti rabbit igg of the then peroxidase labelling of 50 microlitre 1: 10000 dilution, are used after cold PBS washes cell 5 times, add 100 microlitre TMB, hatch after 10 minutes for 37 ℃, use 50 μ L, 1.25M H
2sO
4termination reaction, measures its OD value.The OD of normal control cell
450be 1.21 ± 0.042, the OD of engineering cell
450be that 0.93 ± 0.025(shows as schemed institute 16).The α v of engineering cell expresses and is subject to obvious inhibition.
Above description of test carrier of the present invention can be used in the RNA the Study of Interference of pig genome functions and other pig source cell genes involveds (as: foot and mouth disease, swine fever etc.), stable, lasting RNAi effect can be produced, and fragment insertion point can be estimated for the impact of goal gene.
Claims (6)
1. the rna interference vector based on locus specificity restructuring, it is characterized in that described interference carrier comprises following connected each several part successively: carry out the homologous recombination arm 1 of homologous recombination with certain upstream, single intron region on integrin receptor β 3 genes of pig, human U_6 promoter sequence, polyclone restriction enzyme site, people U6 terminator sequence, the neomycin resistance gene that contains PCMV promotor, carry out the homologous recombination arm 2 of homologous recombination with certain downstream, single intron region on integrin receptor β 3 genes of pig, replication origin gene, amicillin resistance screening-gene and for the linearizing restriction enzyme site of carrier, it is the nucleotide sequence shown in NC_010454.3 that integrin receptor β 3 genes of described pig are arranged in GeneBank accession number.
2. rna interference vector as claimed in claim 1, it is characterized in that it is that extracting from the DNA of pig liver is template that the homologous recombination arm 1 of homologous recombination is carried out in certain upstream, single intron region on described and integrin receptor β 3 genes pig, by using following primer amplification to obtain:
Primers F: 5 '-AAGTCCGGCAGGTGGAGGATTACC-3 '
Primer R:5 '-CCCAGTGCTGGTTATCACATGGC-3 ';
On integrin receptor β 3 genes described and pig, to carry out the homologous recombination arm 2 of homologous recombination be that extracting from the DNA of pig liver is template in certain downstream, single intron region, by using following primer amplification to obtain:
Primers F: 5 '-GGCCATGTGATAACCAGCACTGGGA-3 '
Primer R:5 '-CCCCCTTGTAGTGGACACAGGAGA-3 '.
3. rna interference vector as claimed in claim 1 or 2, is characterized in that the nucleotide sequence of described carrier is as shown in SEQ ID NO.1.
4. a method that builds the rna interference vector described in claim 1-3 any one, is characterized in that comprising the following steps:
(1) structure of homology arm
Take that to extract be template from the DNA of pig liver, design two pairs of primers homologous recombination arm 1 and homologous recombination arm 2 that homologous recombination used that be used for respectively increasing:
Amplification homologous recombination arm 1 primer used is
F:5’-AAGTCCGGCAGGTGGAGGATTACC-3’
R：5’-CCCAGTGCTGGTTATCACATGGC-3’；
Homologous recombination arm 2 primer used is
F：5’-GGCCATGTGATAACCAGCACTGGGA-3’
R：5’-CCCCCTTGTAGTGGACACAGGAGA-3’。
The fragment that clone obtains is connected respectively on PMD20-T carrier, obtains being connected with respectively recombinant vectors PMD20-T-HA1 and the PMD20-T-HA2 of homologous recombination arm 1 and homologous recombination arm 2;
(2) structure of human U_6 promoter, polyclone restriction enzyme site, people U6 terminator sequence
The carrier of take containing human U_6 promoter, polyclone restriction enzyme site, people U6 terminator sequence is template, pcr amplification obtains the fragment that contains human U_6 promoter, polyclone restriction enzyme site, people U6 terminator sequence, the fragment that clone is obtained is connected on PMD20-T carrier, builds and obtains PMD20-T-U6;
(3) structure of positive screening-gene
Neomycin resistance gene NEO and PCMV promotor be take carrier PGT-V1 and are cloned as template, and the amplimer of NEO is:
F:5’-ACACGATGATAAGCTTGCCAC-3’
R：5’-TTAGCTAGCGAACCTCTTCGAGGGACCTAATAACTTC-3’；
The amplimer of PCMV promotor is:
F:5’-ACGGGATCCATATACGCGTTGACATTGATTATTGAC-3’
R：5’-GTTCCCGGGAGAGCTCTGCTTATATAGACCTCCC?AC-3’；
(4) structure of skeleton carrier
Synthetic multiple clone site, and be subcloned on pUC57 carrier, building skeleton carrier pUC57-MCS, multiple clone site sequence is as shown in SEQ ID NO.2;
(5) element assembling
1. after utilizing SpeI on PMD20-T-HA2 carrier and BamHI site enzyme to cut, homologous recombination arm 2 is connected to skeleton carrier pUC57-MCS upper, is configured to carrier pUC57-2;
2. the assembling of PCMV and NEO gene: by after the PCMV being cloned into and the recovery of NEO gene glue, utilize the XmaI restriction enzyme site on gene that two gene splicings are become to PCMV-NEO, PCMV-NEO is cloned into carrier pUC57-2 above, be configured to carrier pUC57-N2;
3. utilize AgeI and the XmaI on SalI and PMD20-T-HA1 and SalI site on PMD20-T-U6, HA1 is cloned into PMD20-T-U6 above, be built into PMD20-T-HA1-U6; Utilize SalI and ClaI site on PMD20-T-HA1-U6 that object fragment HA1-U6 is cloned into pUC57-N2 above, be configured to object carrier PSN-U6.
5. method as claimed in claim 4, is characterized in that the sequence that builds the fragment that contains human U_6 promoter, polyclone restriction enzyme site, people U6 terminator obtaining in step (2) is as shown in SEQ ID NO.3.
6. the application of the rna interference vector described in claim 1-3 any one in building pig source cell RNAi expression vector.
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