CN102250952A - Vector for mediating high-efficiency expression of exogenous gene in cells of mammal and use thereof - Google Patents
Vector for mediating high-efficiency expression of exogenous gene in cells of mammal and use thereof Download PDFInfo
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Abstract
The invention discloses a combined vector for mediating the high-efficiency expression of an exogenous gene in cells of a mammal, which comprises an exogenous gene high-efficiency expression vector pATEW and an artificial transcription factor pcDNA3.1(+)/Hy/GVP4. The exogenous gene high-efficiency expression vector pATEW combines gene expression regulatory elements including a human beta-globin MAR sequence, a hEF-1a genetic transcription regulatory sequence, an after-transcription regulatory element WPRE and the like and an artificial transcription factor combined site sequence. The artificial transcription factor expression vector (pcDNA3.1(+)/Hy/GVP4 is an artificial transcription factor GVP4 which is formed by connecting two parts: four tandem repeats of 12 peptides (DALDDFDLDMLG) in VP16, which serves as the functional structural region of the artificial transcription factor; and the nuclear localization sequence of SV40. By co-transfecting cells of the mammal with the pATEW carrying the exogenous gene and the pcDNA3.1(+)/Hy/GVP4, the high-efficiency expression of the exogenous gene in the cells of the mammal can be realized.
Description
Technical field
The present invention relates to a kind of expression vector, carrier and application thereof that specifically a kind of mediate foreign gene is expressed at mammal cell with high efficient.
Background technology
From (the tissue-type plasminogen activator of tissue-type plasminogen activator in 1987, tPA) since the recombinant protein medicine of producing as the first usefulness mammalian cell expression system went on the market, mammalian cell expression system had occupied dominant position gradually in the production of reorganization pharmaceutical protein.At present, have 65% to 75% to adopt mammalian cell expression system to produce in the recombinant protein medicine of external listing.
The major advantage of mammalian cell expression system is that it can carry out complicated posttranslational modification to protein molecule.These modifications comprise that N-connects the formation that is connected the folding of glycosylation, newborn polypeptide, hydrolysis and disulfide linkage with O-etc.Compare with other expression systems, the expression level of foreign gene in mammalian cell expression system is relatively low.The expression level of foreign gene in mammalian cell be subjected to expression vector transcribe with the stability of the processing of integration site in the host cell gene group of the copy number of translational control element, foreign gene, foreign gene, RNA and mRNA, host cell itself to all multifactor influences such as toxicity of proteic translation modification and secretion capacity and albumen to host cell.
The power of promotor is directly connected to the expression level of foreign gene in host cell, for improving the efficient of transcribing of foreign gene, must select highly active promotor as much as possible for use.CMV promotor (cytomegalovirus promoter) is a promotor the most frequently used in the present eukaryotic expression vector, and it has host cell more widely, also is more intense promotor.But be present in the reticent effect that the interior CpG island of this promotor causes gene easily, the CMV promotor only works in the cell cycle S phase simultaneously.(elongation factor EF) is the protein factor that participates in peptide chain extension in the protein building-up process to elongation factor.The gene of coding EF-1 α is not to be subjected to the house-keeping gene that the cell cycle influences, different steps in the cell cycle all is in high expression level, its locus sequence may contain the transcription regulating nucleotide sequence that some help genetic expression, with the trans-acting factor acting in conjunction of cell, promotes expression of gene.
Transcription factor is a kind ofly can and regulate the specific gene expressed protein in conjunction with DNA.A typical transcription factor is made up of DNA binding domains and functional domain 2 parts in higher eucaryote.The DNA binding domains can be discerned and in conjunction with special dna sequence dna; Functional domain plays transcriptional activation or restraining effect to specific gene.Manual transcription factor perhaps utilizes the various combination of two kinds of structures of natural transcription factor by the structure of people for the natural transcription factor of simulation, creates natural non-existent transcription factor, brings into play the function of natural transcription factor regulate gene expression.Manual transcription factor can improve the efficient of transcribing of foreign gene from transcriptional level, thereby increases the mRNA abundance of foreign gene in the cell, improves the expression of exogenous gene level.
MAR (matrix attachment region) sequence based composition A/T content is greater than 70%, conservative relatively in evolution, contain the MAR distinguished sequence, be called the MAR recognition signal, in the 200bp scope, form by the independent sequence of two portions, different MAR sequence lengths are different, and function also shows difference, and it mainly shows as anti-position effect and regulatory transcription activity.The MAR sequence plays an important role in nucleus and chromosome structure, it with karyomit(e) attached on the nuclear matrix, make the eukaryotic gene group form independently chromosome loop, locate altogether with transcription unit and controlling element in the genome, by promoting to activate the expression of interaction regulatory gene between mixture and the gene.In addition, the MAR sequence is opened the expression of eukaryotic gene with karyomit(e) environment on every side is isolated, make it not be subjected to the influence of negative regulatory element on the karyomit(e), thereby resist genetically modified silence.The MAR sequence is connected on the carrier of expression alien gene, can be in the expression that promotes foreign gene in varying degrees in mammalian cell.
Foreign gene is under the effect of RNA polymerase, and the dna sequence dna in from transcripting start point to the terminating point zone is all transcribed and become RNA.This section comprises that the initial transcript of all introns and exon sequence will be transported in the endochylema then through a series of selectivity processing and splicing, just can carry out correct effectively translation.Wherein, sophisticated mRNA transports out nuclear adjusting and the adjusting in mRNA life-span is effectively to influence the link of foreign gene in mammalian cell expression efficient through processing.(woodchuck hepatitis virus post-transcriptioanl regulatory element WPRE) can promote expression of exogenous gene by the modification to transportation of the caryoplasm of polyadenylic acidization, RNA and translation process to derive from the post-transcriptional control element of alpine marmot hepatitis virus.
Summary of the invention
The objective of the invention is to propose a kind of carrier that mediate foreign gene is expressed at mammal cell with high efficient that is used for.
Invention thinking of the present invention is: it is main the object of the present invention is to provide based on transcriptional level control, post-transcriptional control is the expression vector design philosophy of assisting, reasonable combination people β-globin MAR sequence, manual transcription factor GVP4, people's EF-1 α (human elongation factor 1 alpha, hEF-1 α) mediate foreign gene of gene expression regulation element such as gene transcription regulation sequence and post-transcriptional control element WPRE is at the carrier of mammal cell with high efficient expression, this carrier can effectively overcome position effect, increase promotor efficient, transcribe intensity and increase mRNA and stablize and the exogenous gene high-efficient expressed carrier of exporting controlling element, and use it for and realize foreign gene efficiently expressing at mammalian cell.
Another object of the present invention provides the nucleotide sequence of mediate foreign gene people β-globin MAR sequence, hEF-1 α gene transcription regulation sequence, manual transcription factor binding site sequence and post-transcriptional control element WPRE and manual transcription factor GVP4 in the mammal cell with high efficient expression vector.
The 3rd purpose of the present invention provides the construction process of mediate foreign gene at the mammal cell with high efficient expression vector.
The 4th purpose of the present invention provides the application of mediate foreign gene at the mammal cell with high efficient expression vector.
For achieving the above object, the present invention is by the following technical solutions:
The carrier that a kind of mediate foreign gene is expressed at mammal cell with high efficient, described carrier comprises people β-globin MAR, hEF-1 α gene transcription regulation element, manual transcription factor binding site, manual transcription factor GVP4 and post-transcriptional control element WPRE;
The sequence of described people β-globin MAR is shown in SEQ ID No.1;
Described hEF-1 α gene transcription regulation element comprises the nucleotide sequence of the hEF-1 α 5 ' end shown in SEQ ID No.2 and the nucleotide sequence of the 3 ' end of the hEF-1 α shown in the SEQ ID No.3;
The sequence of described manual transcription factor binding site is shown in SEQ ID No.4;
The sequence of described post-transcriptional control element WPRE is shown in SEQ ID No.5;
The nucleotide sequence of described manual transcription factor GVP4 is shown in SEQ ID No.6.
The carrier that described mediate foreign gene is expressed at mammal cell with high efficient, wherein,
Described people β-globin MAR, hEF-1 α gene transcription regulation element, post-transcriptional control element WPRE and manual transcription factor binding site are combined into exogenous gene high-efficient expressed carrier pATEW;
Described manual transcription factor GVP4 has made up manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4.
The nucleotide sequence of the hEF-1 α 5 ' end of described hEF-1 α gene transcription regulation element be positioned at mediation outside
The upstream of source gene, the nucleotide sequence of hEF-1 α 3 ' end is positioned at the downstream of the foreign gene of mediation.
Described manual transcription factor binding site is positioned at the upstream of the nucleotide sequence of hEF-1 α 5 ' end.
Described people β-globin MAR is positioned at the upstream of manual transcription factor binding site.
Described post-transcriptional control element WPRE is positioned at the upstream of the nucleotide sequence of hEF-1 α 3 ' end.
Described exogenous gene high-efficient expressed carrier pATEW as shown in Figure 1; Described manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4 as shown in Figure 2.
The construction of carrier that a kind of above-mentioned mediate foreign gene is expressed at mammal cell with high efficient, described method comprises the steps:
(1) be carrier framework with pcDNA3.1 (+), replace the CMV promotor of pcDNA3.1 (+) with hEF-1 α gene transcription regulation element, the manual transcription factor binding site sequence that contains 10 GaL4 binding sites is inserted in upstream at the nucleotide sequence of hEF-1 α 5 ' end, people β-globin MAR is inserted in upstream in manual transcription factor binding site sequence, post-transcriptional control element WPRE is inserted in upstream at the nucleotide sequence of hEF-1 α 3 ' end, is built into exogenous gene high-efficient expressed carrier pATEW;
(2) be carrier framework with pcDNA3.1 (+), replace the neomycin resistance gene Neo (R) of pcDNA3.1 (+) with hygromycin gene Hygro (R), the downstream of CMV promotor insert coding by yeast transcription factor GAL4 among 1-147 amino acids and the hsv transcription activator V166 4 tandem repetitive sequences of 12 peptides (DALDDFDLDMLG) forms the nucleotide sequence of manual transcription factor GVP4, structure manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4.
The application of the expression vector that aforesaid method makes up in mediation encoding antibody, Fc fusion rotein, cytokine, thrombin and of the expression of somatomedin foreign gene at mammalian cell.
A kind of carrier that utilizes method for preparing is in the mammal cell with high efficient expression method, and step is as follows:
(1) will encode the respectively cDNA of enhanced green fluorescence protein EGFP, tPA, uPA proUK and Enbrel is inserted into the downstream of the nucleotide sequence of hEF-1 α 5 ' end among the described pATEW, is built into carrier pATEW/EGFP, pATEW/tPA, pATEW/proUK and the pATEW/Enbrel of the different exogenous gene expressions of mediation;
(2) mol ratio cotransfection Chinese hamster ovary celI or HEK293 cell such as respectively pATEW/EGFP, pATEW/tPA, pATEW/proUK and pATEW/Enbrel and manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4 are pressed;
(3) clone of screening efficiently expressing exogenous gene.
The invention has the beneficial effects as follows:
(1) the present invention is the structure that gene expression regulation unit construction such as people β-globin MAR sequence, manual transcription factor GVP4, hEF-1 α gene transcription regulation sequence and post-transcriptional control element WPRE is applied to mammalian cell expression vector by molecular biology method.
(2) constructed mammalian cell expression vector can overcome output efficiency and the stability that position effect, increase promotor efficient and enhancing are transcribed intensity, strengthened mRNA, mediate foreign gene efficiently expressing in mammalian cell effectively.
The invention will be further described below in conjunction with drawings and Examples.
Description of drawings
Fig. 1 is the structural representation of exogenous gene high-efficient expressed carrier pATEW.
Fig. 2 is the structural representation of artificial transcription factor expression carrier pcDNA3.1 (+)/Hy/GVP4.
Fig. 3 is the structural representation of exogenous gene expression carrier pATEW/EGFP, pATEW/tPA, pATEW/proUK and pATEW/Enbrel.
The pATEW mediation EGFP that Fig. 4 observes for inverted microscope expresses (A) in Chinese hamster ovary celI and macroscopic pATEW mediation EGFP expresses (B) in HEK 293 cells.
Fig. 5 is the flow cytometry figure of pATEW/EGFP and pcDNA3.1/EGFP mediation EGFP expression effect in Chinese hamster ovary celI.
The effect that Fig. 6 expresses in Chinese hamster ovary celI for SDS-PAGE express foreign gene efficient expression vector pATEW mediation Enbrel.(M: standard molecular weight Maker; E: commercialization Enbre product 1-8: the serum-free feeding culture is expressed the supernatant sample of Enbrel Chinese hamster ovary celI)
Embodiment
The clone of the MAR sequence of embodiment 1 people β-globin
From HEK293 cell extraction genomic dna, people β-globin MAR sequence according to the GenBank report, wherein contain one section Alu sequence and in the people's gene group, should be tumor-necrosis factor glycoproteins, adopt segmentation clone's way, method clone 1-2804bp with PCR, with artificial synthetic method Synthetic 2 804-2998bp, both being spliced acquisition length is people β-globin MAR sequence of 2998bp again.Synthetic HUMAR1, HUMAR2 primer, be used to the to increase fragment of 2804bp, amplification condition is: 94 ℃ of sex change 4min, 94 ℃ of 1min, 68 ℃ of 1min, 72 ℃ of 2min40sec totally 30 circulations, 72 ℃ are extended 10min, 4 ℃ of coolings, used archaeal dna polymerase are LA TaqDNA polysaccharase.
Primer HUMAR1 sequence is shown in SEQ ID No.7.
Primer HUMAR2 sequence is shown in SEQ ID No.8.
The clone of embodiment 2hEF-1 α gene transcription regulation element
The following primer of sequences Design according to the report of Genbank:
Primer HuEF1 sequence is shown in SEQ ID No.9.
Primer HuEF2 sequence is shown in SEQ ID No.10.
Primer HuEF3 sequence is shown in SEQ ID No.11.
Primer HuEF4 sequence is shown in SEQ ID No.12.
Comprise the nucleotide sequence that the hEF-1 α 5 ' of promotor and introne 1 holds with primer HuEF1 and HuEF2 amplification 4093bp, amplified production contains NheI and MluI restriction enzyme site; The nucleotide sequence of the hEF-1 α 3 ' end of primer HuEF3 and HuEF4 amplification 4018bp, amplified production contains XbaI and BspEI restriction enzyme site.Adopt the LA Taq archaeal dna polymerase of precious biotech firm.Amplification condition is 94 ℃ of 5min, 94 ℃ of 30sec, and 60 ℃ of 30sec, 72 ℃ of 4min, 30 circulations, 72 ℃ are extended 10min, and amplified production 1% agarose gel reclaims, and is connected with the T carrier.Enzyme is cut with sequencing result and is shown that the sequence that is obtained is correct sequence.
The clone of embodiment 3 manual transcription factor binding site nucleotide sequences
Clone for convenience, primer UASPacI (SEQ ID No.13), UASAgeI1 (SEQ ID No.14), UASAgeI2 (SEQ ID No.15), UASHpaI (SEQ ID No.16) 5 ' end have designed PacI, AgeI, HpaI restriction enzyme site respectively, contain the manual transcription factor binding site sequence of 5 GaL4 binding sites from plasmid PG5luc (Promega product) amplification with these two primers.With the Pybest enzymatic amplification of TAKARA high-fidelity, reclaim the fragment of about 150bp.Connect into pcDNA3.1 (+)/MCS carrier by PacI, AgeI, connect into pcDNA3.1 (+)/UAS, the correct back of order-checking called after pcDNA3.1 (+)/2UAS by AgeI, HpaI.The clone of embodiment 4 post-transcriptional control element WPRE sequences
(woodchuck hepatitis virus, the WHV) gene order of X protein have been cloned the WPRE of 1094-1694bp, and enzyme is cut and identified and order-checking is identified correct according to the coding alpine marmot hepatitis virus of GenBank report.
The sequence of described post-transcriptional control element WPRE is shown in SEQ ID No.5;
The structure of embodiment 5 exogenous gene high-efficient expressed carrier pATEW
Adopt conventional Protocols in Molecular Biology method to replace the CMV promoter sequence of pcDNA3.1 (+)/2UAS carrier earlier with the nucleotide sequence of 5/ end of hEF-1 α and 3/ end; Again people β-globin MAR sequence is connected into the upstream of manual transcription factor binding site sequence; Post-transcriptional control element WPRE is inserted in the upstream that is preferably in hEF-1 α 3 ' ending regulating sequence, makes up exogenous gene high-efficient expressed carrier pATEW.It is correct to cut the constructed pATEW carrier of evaluation through enzyme, and carrier structure as shown in Figure 1.
The clone of the nucleotide sequence of embodiment 6 coding manual transcription factor GVP4
Design two primer GAL1 (SEQ ID No.17), GAL2 (SEQ ID No.18), clone for convenience, 5 ' end of primer 1 has designed EcoRI and NheI restriction enzyme site, and designed KOZAKA sequence GCC GCC ACC ATG, 5 ' end of primer 2 has designed the KpnI restriction enzyme site, bring into play function in the nucleus for manual transcription factor can be entered, in primer 2, designed nuclear localization sequence: ATC TAC CTT TCT CTT CTT TTT TGG.With these two primers from plasmid pBind amplification GAL4 sequence.With the Pybest enzyme of TAKARA high-fidelity, reclaim the fragment of about 450bp.Be connected into PUC19 carrier with KpnI by EcoRI, the correct back of order-checking called after GAL4/PUC19.
Proteic one section 11 peptide sequence of bibliographical information hsv transcription activator VP16 (DALDDFDLDML) has the proteic mobilizing function of VP16, and 4 series connection multiple 11 peptide sequences have stronger activation effect; 2 of 8 amino acid whose small peptides (DFDLDMLG) series connection repeat among the VP16, have also shown stronger transcriptional activity.The present invention adopts four series connection of 12 peptides (DALDDFDLDMLG) to repeat as manual transcription factor mobilizing function structural domain, design 4 primer VP1 (SEQ ID No.19), VP2 (SEQ ID No.20), VP3 (SEQ ID No.21), VP4 (SEQ ID No.22), as 4 series connection of synthetic multiple, 12 peptides.Nuclear localization sequence (NLS) with SV40 couples together two portions, has made up manual transcription factor GVP4 (SEQ ID No.6).
With 50 μ l, 1 * PCR reaction buffer (pyrobest buffer TAKARA product) VP1 and each 50pmol of VP2 are dissolved in the 0.5ml centrifuge tube, place a beaker that fills with boiling water, make the boiling water naturally cooling at room temperature in the beaker, make oligonucleotide be annealed into two strands, two ends form BamHI and KpnI restriction enzyme site respectively; Cutting processing GAL4/PUC19 recovery back with BamHI with the KpnI enzyme is connected with the linker that VP1, VP2 form.After the same processing of VP3, VP4, be connected with the carrier of cutting behind the GVP2/PUC19 with BamHI, HindIII enzyme.Enzyme is cut with sequencing result and is shown that the sequence that is obtained is correct sequence.
The structure of embodiment 7 manual transcription factor expression vectors
Cut with NheI, HindIII enzyme, reclaim the fragment GVP4 about 600bp, cut carrier pcDNA3.1/Hygro (+) with NheI, HindIII enzyme equally, reclaim carrier and be connected with GVP4.Connect correct called after pcDNA3.1/Hy/GVP4, carrier structure as shown in Figure 2.
The foreign gene of embodiment 8pATEW mediation efficiently expresses in mammalian cell
Be connected into mammal cell with high efficient expression vector pATEW by will encode the respectively foreign gene of EGFP, tPA, proUK and Enbrel of multiple clone site, mammalian cell expression vector pATEW/EGFP, pATEW/tPA, pATEW/proUK and the pATEW/Enbrel of difference construction expression EGFP, tPA, proUK and Enbrel, the structure of each expression vector as shown in Figure 3.
Embodiment 9 efficient expression vector cotransfection mammalian cells
Respectively with pATEW/EGFP, pATEW/tPA, pATEW/proUK and pATEW/Enbrel and manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4 by waiting mole to mix usefulness transfection reagent Lepofectamine2000 cotransfection Chinese hamster ovary celI and HEK293 cell.
The screening and the clone of embodiment 10 exogenous gene high-efficient expressed cells
Through the positive Chinese hamster ovary celI of the transfection of G418 pressurization screening and HEK293 cell or with inverted microscope, visual inspection EGFP expression intensity, as shown in Figure 4.Cells were tested by flow cytometry EGFP expression intensity, pATEW mediation EGFP expression efficiency and the expression efficiency of commercialization carrier pcDNA3.1 (+) mediation EGFP in Chinese hamster ovary celI in Chinese hamster ovary celI improves about 29.3 times, as shown in Figure 5.Or the clone of determining exogenous gene expression level, screening efficiently expressing exogenous gene in conjunction with the mensuration and the cell counting of exogenous object protein with limiting dilution assay clone.Wherein, the mensuration of tPA and proUK adopts external scleroproein agar plate solusphere method; The ELISA method is adopted in the detection of Enbrel.(Innovatis Germany) carries out viable count to adopt Cedex AS20 cell density and vigor automatic analysis system.Having obtained proUK stably express level respectively through colony screening is that 20-25pg/cell/day, tPA stably express level are that 25-30pg/cell/day and Enbrel stably express level are the clone of 50-60pg/cell/day.SDS-PAGE analyzes the supernatant sample that the serum-free feeding culture is expressed the Enbrel Chinese hamster ovary celI, presents tangible Enbrel in the position identical with commercialization Enbrel product and colour band, as shown in Figure 6.
Claims (10)
1. the mediate foreign gene carrier of expressing at mammal cell with high efficient, it is characterized in that: described carrier comprises people β-globin MAR, hEF-1 α gene transcription regulation element, manual transcription factor binding site, manual transcription factor GVP4 and post-transcriptional control element WPRE;
The sequence of described people β-globin MAR is shown in SEQ ID No.1;
Described hEF-1 α gene transcription regulation element comprises the nucleotide sequence of the hEF-1 α 5 ' end shown in SEQ ID No.2 and the nucleotide sequence of the 3 ' end of the hEF-1 α shown in the SEQ ID No.3;
The sequence of described manual transcription factor binding site is shown in SEQ ID No.4;
The sequence of described post-transcriptional control element WPRE is shown in SEQ ID No.5;
The nucleotide sequence of described manual transcription factor GVP4 is shown in SEQ ID No.6.
2. the carrier that mediate foreign gene according to claim 1 is expressed at mammal cell with high efficient is characterized in that:
Described people β-globin MAR, hEF-1 α gene transcription regulation element, post-transcriptional control element WPRE and manual transcription factor binding site are combined into exogenous gene high-efficient expressed carrier pATEW;
Described manual transcription factor GVP4 has made up manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4.
3. the carrier that mediate foreign gene according to claim 2 is expressed at mammal cell with high efficient, it is characterized in that: the nucleotide sequence of the hEF-1 α 5 ' end of described hEF-1 α gene transcription regulation element is positioned at the upstream of the foreign gene of mediation, and the nucleotide sequence of hEF-1 α 3 ' end is positioned at the downstream of the foreign gene of mediation.
4. the carrier that mediate foreign gene according to claim 3 is expressed at mammal cell with high efficient is characterized in that: described manual transcription factor binding site is positioned at the upstream of the nucleotide sequence of hEF-1 α 5 ' end.
5. the carrier that mediate foreign gene according to claim 4 is expressed at mammal cell with high efficient, it is characterized in that: described people β-globin MAR is positioned at the upstream of manual transcription factor binding site.
6. the carrier that mediate foreign gene according to claim 5 is expressed at mammal cell with high efficient is characterized in that: described post-transcriptional control element WPRE is positioned at the upstream of the nucleotide sequence of hEF-1 α 3 ' end.
7. according to the carrier of any described mediate foreign gene in the claim 2 to 6 in the mammal cell with high efficient expression, it is characterized in that: described exogenous gene high-efficient expressed carrier pATEW as shown in Figure 1; Described manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4 as shown in Figure 2.
8. the construction of carrier expressed at mammal cell with high efficient of the described mediate foreign gene of claim 1, it is characterized in that: described method comprises the steps:
(1) be carrier framework with pcDNA3.1 (+), replace the CMV promotor of pcDNA3.1 (+) with hEF-1 α gene transcription regulation element, the manual transcription factor binding site sequence that contains 10 GaL4 binding sites is inserted in upstream at the nucleotide sequence of hEF-1 α 5 ' end, people β-globin MAR is inserted in upstream in manual transcription factor binding site sequence, post-transcriptional control element WPRE is inserted in upstream at the nucleotide sequence of hEF-1 α 3 ' end, is built into exogenous gene high-efficient expressed carrier pATEW;
(2) be carrier framework with pcDNA3.1 (+), replace the neomycin resistance gene Neo (R) of pcDNA3.1 (+) with hygromycin gene Hygro (R), the downstream of CMV promotor insert coding by yeast transcription factor GAL4 among 1-147 amino acids and the hsv transcription activator VP16 4 tandem repetitive sequences of 12 peptides (DALDDFDLDMLG) forms the nucleotide sequence of manual transcription factor GVP4, structure manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4.
9. the application of the expression vector of claim 8 structure in mediation encoding antibody, Fc fusion rotein, cytokine, thrombin and of the expression of somatomedin foreign gene at mammalian cell.
10. a carrier that utilizes claim 8 preparation is characterized in that in the mammal cell with high efficient expression method step is as follows:
(1) will encode the respectively cDNA of enhanced green fluorescence protein EGFP, tPA, uPA proUK and Enbrel is inserted into the downstream of the nucleotide sequence of hEF-1 α 5 ' end among the described pATEW, is built into carrier pATEW/EGFP, pATEW/tPA, pATEW/proUK and the pATEW/Enbrel of the different exogenous gene expressions of mediation;
(2) mol ratio cotransfection Chinese hamster ovary celI or HEK293 cell such as respectively pATEW/EGFP, pATEW/tPA, pATEW/proUK and pATEW/Enbrel and manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4 are pressed;
(3) clone of screening efficiently expressing exogenous gene.
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| CN105039411A (en) * | 2015-08-27 | 2015-11-11 | 新乡医学院 | Attached type lentiviral vector as well as preparation method and application thereof |
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| CN102703503A (en) * | 2012-06-01 | 2012-10-03 | 新乡医学院 | Human and mammal cell attachment expression vector and application thereof |
| CN102703503B (en) * | 2012-06-01 | 2013-08-28 | 新乡医学院 | Human and mammal cell attachment expression vector and application thereof |
| CN102775499A (en) * | 2012-08-01 | 2012-11-14 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g64730 gene |
| CN102775499B (en) * | 2012-08-01 | 2013-11-13 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g64730 gene |
| CN105039411A (en) * | 2015-08-27 | 2015-11-11 | 新乡医学院 | Attached type lentiviral vector as well as preparation method and application thereof |
| CN105039411B (en) * | 2015-08-27 | 2019-01-18 | 新乡医学院 | Attached type slow virus carrier, preparation method and application |
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