CN102250952B - The carrier that mediate foreign gene is expressed at mammal cell with high efficient and application thereof - Google Patents
The carrier that mediate foreign gene is expressed at mammal cell with high efficient and application thereof Download PDFInfo
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Abstract
The invention discloses the combination carrier that mediate foreign gene is expressed at mammal cell with high efficient, this combination carrier comprises exogenous gene high-efficient expressed carrier pATEW and manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4.Does is exogenous gene high-efficient expressed carrier pATEW combined with people β-globin? gene expression regulation element and the manual transcription factor binding site sequences such as MAR sequence, hEF-1 α gene transcription regulation sequence, posttranscriptional regulatory element WPRE; Manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4 expresses the manual transcription factor GVP4 be formed by connecting as the functional domain of manual transcription factor and nuclear localization sequence two portions of SV40 by 4 tandem sequence repeats of 12 peptides (DALDDFDLDMLG) in VP16.By pATEW and pcDNA3.1 (+) of foreign gene-carrying/Hy/GVP4 cotransfection mammalian cell, the high expression of foreign gene in mammalian cell can be realized.
Description
Technical field
The present invention relates to a kind of expression vector, specifically a kind of mediate foreign gene carrier of expressing at mammal cell with high efficient and application thereof.
Background technology
From tissue-type plasminogen activator (tissue-typeplasminogenactivator in 1987, tPA), since going on the market with the recombinant protein medicine that mammalian cell expression system is produced as first, mammalian cell expression system occupies dominant position gradually in the production of restructuring pharmaceutical protein.At present, 65% to 75% is had to adopt mammalian cell expression system to produce in the external recombinant protein medicine gone on the market.
The major advantage of mammalian cell expression system is that it can carry out complicated posttranslational modification to protein molecule.These are modified and comprise N-and connect and to be connected glycosylation, the folding of nascent polypeptide with O-, to be hydrolyzed and the formation etc. of disulfide linkage.Compared 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 to transcribe with the stability of the processing of the copy number of translational control element, foreign gene, the integration site of foreign gene in host cell gene group, RNA and mRNA, host cell itself the translation modification of albumen and secretion capacity and albumen the impact of the factors such as the toxicity of host cell by expression vector.
The power of promotor is directly connected to the expression level of foreign gene in host cell, for improving the transcriptional efficiency of foreign gene, must select highly active promotor as much as possible.CMV promoter (cytomegaloviruspromoter) is promotor the most frequently used in current eukaryotic expression vector, and it has host cell more widely, is also stronger promotor.But be present in the reticent effect that this promotor Nei CpG island easily causes gene, CMV promoter only works in the S phase of cell cycle simultaneously.Elongation factor (elongationfactor, EF) are the protein factors participating in peptide chain extension in protein building-up process.The gene of coding EF-1 α is not by the house-keeping gene of Cell cycle influences, all high expression level is in the different steps of cell cycle, its locus sequence may be conducive to the transcription regulating nucleotide sequence of genetic expression containing some, with the trans-acting factor acting in conjunction of cell, promote the expression of gene.
Transcription factor a kind ofly in conjunction with DNA and can regulate the protein of neural specific gene expression.In higher eucaryote, a typical transcription factor is made up of DNA binding domains and functional domain 2 part.DNA binding domains can identify and in conjunction with special DNA sequence dna; Functional domain plays transcriptional activation or restraining effect to specific gene.Manual transcription factor is the structure of the simulation native transcription factor by people, or utilizes the various combination of two kinds of structures of the native transcription factor, creates natural non-existent transcription factor, plays the function of native transcription cytokine regulatory genetic expression.Manual transcription factor can improve the transcriptional efficiency of foreign gene from transcriptional level, thus increases the mRNA abundance of intraor extracellular source gene, improves the expression level of foreign gene.
MAR (matrixattachmentregion) series composition A/T content is greater than 70%, relatively conservative in evolution, containing MAR distinguished sequence, be called MAR recognition signal, be made up of the independent sequence of two portions within the scope of 200bp, different MAR sequence length is different, and function also shows difference, and its main manifestations is that anti-position effect and regulatory transcription are active.MAR sequence plays an important role in nucleus and chromosome structure, karyomit(e) is attached on nuclear matrix by it, eukaryotic gene group is made to form independently chromosome loop, locate altogether with the transcription unit in genome and controlling element, by promoting the expression of the interphase interaction regulatory gene activating mixture and gene.In addition, the karyomit(e) environment of the expression of eukaryotic gene and surrounding is opened by MAR sequence, makes it not by the impact of negative regulatory element on karyomit(e), thus resists genetically modified silence.MAR sequence is connected on the carrier of expression alien gene, can in the expression promoting foreign gene in varying degrees in mammalian cell.
Foreign gene, under the effect of RNA polymerase, is all transcribed from transcripting start point to the DNA sequence dna in terminating point region and is become RNA.The primary transcript that this section comprises all introns and exon sequence through a series of selectivity processing and splicing, then will be transported in endochylema, just can carry out correct effective translation.Wherein, it is effectively affect the link of foreign gene in mammalian cell expression efficiency that the mRNA through processing maturation transports out the adjustment in nuclear adjustment and mRNA life-span.The posttranscriptional regulatory element (woodchuckhepatitisviruspost-transcriptioanlregulatoryele ment, WPRE) deriving from alpine marmot hepatitis virus can by promoting the expression of foreign gene to the nucleocytoplasmic transport of Polyadenylation, RNA and the modification of translation process.
Summary of the invention
The object of the invention is to propose a kind of carrier of expressing at mammal cell with high efficient for mediate foreign gene.
Invention thinking of the present invention is: it is main for the object of the present invention is to provide based on transcriptional level control, post-transcriptional control is auxiliary expression vector design philosophy, reasonable combination people β-globinMAR sequence, manual transcription factor GVP4, people's EF-1 α (humanelongationfactor1alpha, hEF-1 α) mediate foreign gene of the gene expression regulation element such as gene transcription regulation sequence and the posttranscriptional regulatory element WPRE carrier of expressing at mammal cell with high efficient, this carrier effectively can overcome position effect, increase promotor efficiency, Intensity of Transcription of Endothelial and increase mRNA stablize and export the exogenous gene high-efficient expressed carrier of controlling element, and use it for and realize the high expression of foreign gene at mammalian cell.
Another object of the present invention is to provide the nucleotide sequence of mediate foreign gene people β-globinMAR sequence, hEF-1 α gene transcription regulation sequence, manual transcription factor binding site sequence and posttranscriptional regulatory element WPRE and manual transcription factor GVP4 in High Expression Vectors in Mammalian Cells.
3rd object of the present invention is to provide the construction process of mediate foreign gene at High Expression Vectors in Mammalian Cells.
4th object of the present invention is to provide the application of mediate foreign gene at High Expression Vectors in Mammalian Cells.
For achieving the above object, the present invention is by the following technical solutions:
The carrier that mediate foreign gene is expressed at mammal cell with high efficient, described carrier comprises people β-globinMAR, hEF-1 α gene transcription regulation element, manual transcription factor binding site, manual transcription factor GVP4 and posttranscriptional regulatory element WPRE;
The sequence of described people β-globinMAR is as shown in SEQIDNo.1;
Described hEF-1 α gene transcription regulation element comprises nucleotide sequence that the hEF-1 α 5 ' as shown in SEQIDNo.2 holds and the nucleotide sequence that the hEF-1 α 3 ' shown in SEQIDNo.3 holds;
The sequence of described manual transcription factor binding site is as shown in SEQIDNo.4;
The sequence of described posttranscriptional regulatory element WPRE is as shown in SEQIDNo.5;
The nucleotide sequence of described manual transcription factor GVP4 is as shown in SEQIDNo.6.
The carrier that described mediate foreign gene is expressed at mammal cell with high efficient, wherein,
Described people β-globinMAR, hEF-1 α gene transcription regulation element, posttranscriptional regulatory element WPRE and manual transcription factor binding site are combined into exogenous gene high-efficient expressed carrier pATEW;
Described manual transcription factor GVP4 constructs manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4.
The nucleotide sequence that the hEF-1 α 5 ' of described hEF-1 α gene transcription regulation element holds is positioned at outside mediation
The upstream of source gene, the nucleotide sequence that hEF-1 α 3 ' holds 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 that hEF-1 α 5 ' holds.
Described people β-globinMAR is positioned at the upstream of manual transcription factor binding site.
Described posttranscriptional regulatory element WPRE is positioned at the upstream of the nucleotide sequence that hEF-1 α 3 ' holds.
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.
A construction process for the carrier that above-mentioned mediate foreign gene is expressed at mammal cell with high efficient, described method comprises the steps:
(1) with pcDNA3.1 (+) for carrier framework, the CMV promoter of pcDNA3.1 (+) is replaced with hEF-1 α gene transcription regulation element, the manual transcription factor binding site sequence containing 10 GaL4 binding sites is inserted in the upstream of the nucleotide sequence held at hEF-1 α 5 ', people β-globinMAR is inserted in the upstream of manual transcription factor binding site sequence, at the upstream insertion posttranscriptional regulatory element WPRE of the nucleotide sequence that hEF-1 α 3 ' holds, be built into exogenous gene high-efficient expressed carrier pATEW;
(2) with pcDNA3.1 (+) for carrier framework, the neomycin resistance gene Neo (R) of pcDNA3.1 (+) is replaced with hygromycin gene Hygro (R), insert the nucleotide sequence of 4 tandem repetitive sequences composition manual transcription factor GVP4 of 12 peptides (DALDDFDLDMLG) in 1-147 amino acids and hsv transcription activator V166 in coding yeast transcription factor GAL4 in the downstream of CMV promoter, build manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4.
The application of the expression vector that aforesaid method builds in mediation encoding antibody, Fc fusion rotein, cytokine, thrombin and the expression of somatomedin foreign gene at mammalian cell.
The carrier utilizing aforesaid method to prepare is in a mammal cell with high efficient expression method, and step is as follows:
(1) respectively the cDNA of coding enhanced green fluorescence protein EGFP, tPA, uPA proUK and Enbrel is inserted into the downstream of the nucleotide sequence that hEF-1 α 5 ' holds in described pATEW, is built into carrier pATEW/EGFP, pATEW/tPA, pATEW/proUK and pATEW/Enbrel of mediating different exogenous gene expression;
(2) respectively pATEW/EGFP, pATEW/tPA, pATEW/proUK and pATEW/Enbrel and manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4 are pressed equimolar ratio cotransfection Chinese hamster ovary celI or HEK293 cell;
(3) clone of efficiently expressing exogenous gene is screened.
The invention has the beneficial effects as follows:
(1) the present invention is the structure by molecular biology method, the gene expression regulation unit construction such as people β-globinMAR sequence, manual transcription factor GVP4, hEF-1 α gene transcription regulation sequence and posttranscriptional regulatory element WPRE being applied to mammalian cell expression vector.
(2) mammalian cell expression vector constructed by effectively can overcome position effect, increases promotor efficiency and strengthen Intensity of Transcription of Endothelial, the output efficiency strengthening mRNA and stability, the high expression of mediate foreign gene in mammalian cell.
Below in conjunction with drawings and Examples, the invention will be further described.
Accompanying drawing explanation
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.
Fig. 4 is that the pATEW mediation EGFP of inverted microscope observation expresses (A) in Chinese hamster ovary celI and macroscopic pATEW mediates EGFP at HEK293 cells (B).
Fig. 5 is the flow cytometry figure that pATEW/EGFP and pcDNA3.1/EGFP mediates EGFP expression effect in Chinese hamster ovary celI.
Fig. 6 is the effect that SDS-PAGE express foreign gene efficient expression vector pATEW mediation Enbrel expresses in Chinese hamster ovary celI.(M: standard molecular weight Maker; E: commercialization Enbre product 1-8: serum-free feeding culture expresses the Supernatant samples of EnbrelCHO cell)
Embodiment
The clone of the MAR sequence of embodiment 1 people β-globin
From HEK293 cell extraction genomic dna, according to the people β-globinMAR sequence of GenBank report, wherein in human genome, should be tumor-necrosis factor glycoproteins containing one section of Alu sequence, adopt the way of cDNA clones, with the method clone 1-2804bp of PCR, with the method synthesis 2804-2998bp of synthetic, then both are spliced the people β-globinMAR sequence that acquisition length is 2998bp.Synthesis HUMAR1, HUMAR2 primer, for the fragment of the 2804bp that increases, amplification condition is: 94 DEG C of sex change 4min, 94 DEG C of 1min, 68 DEG C of 1min, 72 DEG C of 2min40sec totally 30 circulations, and 72 DEG C extend 10min, 4 DEG C of coolings, archaeal dna polymerase used is LATaqDNA polysaccharase.
Primer HUMAR1 sequence is as shown in SEQIDNo.7.
Primer HUMAR2 sequence is as shown in SEQIDNo.8.
The clone of embodiment 2hEF-1 α gene transcription regulation element
According to the following primer of the sequences Design of the report of Genbank:
Primer HuEF1 sequence is as shown in SEQIDNo.9.
Primer HuEF2 sequence is as shown in SEQIDNo.10.
Primer HuEF3 sequence is as shown in SEQIDNo.11.
Primer HuEF4 sequence is as shown in SEQIDNo.12.
With the nucleotide sequence that the hEF-1 α 5 ' that primer HuEF1 and HuEF2 amplification 4093bp comprises promotor and introne 1 holds, amplified production is containing NheI and MluI restriction enzyme site; The nucleotide sequence that the hEF-1 α 3 ' that primer HuEF3 and HuEF4 increases 4018bp holds, amplified production is containing XbaI and BspEI restriction enzyme site.Adopt the LATaqDNA polysaccharase of precious biotech firm.Amplification condition is 94 DEG C of 5min, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 4min, 30 circulations, and 72 DEG C extend 10min, and amplified production 1% agarose gel reclaims, and is connected with carrier T.Enzyme is cut and is shown that obtained sequence is correct sequence with sequencing result.
The clone of embodiment 3 manual transcription factor binding site nucleotide sequence
Conveniently clone, primer UASPacI (SEQIDNo.13), UASAgeI1 (SEQIDNo.14), UASAgeI2 (SEQIDNo.15), UASHpaI (SEQIDNo.16) 5 ' end devises PacI, AgeI, HpaI restriction enzyme site respectively, contains the manual transcription factor binding site sequence of 5 GaL4 binding sites with these two primers from plasmid PG5luc (Promega product) amplification.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 by AgeI, HpaI, called after pcDNA3.1 (+)/2UAS after order-checking is correct.The clone of embodiment 4 posttranscriptional regulatory element WPRE sequence
According to the gene order of coding alpine marmot hepatitis virus (woodchuckhepatitisvirus, the WHV) X protein of GenBank report, cloned the WPRE of 1094-1694bp, enzyme cuts qualification and order-checking qualification is correct.
The sequence of described posttranscriptional regulatory element WPRE is as shown in SEQIDNo.5;
The structure of the exogenous gene high-efficient expressed carrier pATEW of embodiment 5
Conventional Protocols in Molecular Biology method is adopted first to replace the CMV promoter sequence of pcDNA3.1 (+)/2UAS carrier with 5/ end of hEF-1 α and the nucleotide sequence of 3/ end; Again people β-globinMAR sequence is connected into the upstream of manual transcription factor binding site sequence; Preferably insert posttranscriptional regulatory element WPRE in the upstream of hEF-1 α 3 ' ending regulating sequence, build exogenous gene high-efficient expressed carrier pATEW.The pATEW carrier cut constructed by qualification through enzyme is correct, and carrier structure as shown in Figure 1.
Embodiment 6 is encoded the clone of nucleotide sequence of manual transcription factor GVP4
Design two primer GAL1 (SEQIDNo.17), GAL2 (SEQIDNo.18), conveniently clone, 5 ' end of primer 1 devises EcoRI and NheI restriction enzyme site, and devise KOZAKA sequence GCCGCCACCATG, 5 ' end of primer 2 devises KpnI restriction enzyme site, enter in nucleus to enable manual transcription factor and play function, in primer 2, devise nuclear localization sequence: ATCTACCTTTCTCTTCTTTTTTGG.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 by EcoRI with KpnI, called after GAL4/PUC19 after order-checking is correct.
One section of 11 peptide sequence (DALDDFDLDML) of bibliographical information hsv transcription activator VP16 albumen has the mobilizing function of VP16 albumen, and 11 peptide sequences of 4 tandem sequence repeats have stronger activation effect; In VP16,2 tandem sequence repeats of 8 amino acid whose small peptides (DFDLDMLG), also show stronger transcriptional activity.The present invention adopts four tandem sequence repeats of 12 peptides (DALDDFDLDMLG) as manual transcription factor mobilizing function structural domain, design 4 primer VP1 (SEQIDNo.19), VP2 (SEQIDNo.20), VP3 (SEQIDNo.21), VP4 (SEQIDNo.22), as 12 peptides of synthetic 4 tandem sequence repeats.With the nuclear localization sequence (NLS) of SV40, two portions are coupled together, construct manual transcription factor GVP4 (SEQIDNo.6).
With 50 μ l1 × PCR reaction buffers (pyrobestbufferTAKARA product), each for VP1 and VP2 50pmol is dissolved in 0.5ml centrifuge tube, be placed in the beaker that fills with boiling water, make the boiling water naturally cooling at room temperature in beaker, make oligonucleotide be annealed into double-strand, two ends form BamHI and KpnI restriction enzyme site respectively; Cut after process GAL4/PUC19 reclaims with BamHI with KpnI enzyme and be connected with the linker that VP1, VP2 are formed.After VP3, VP4 process equally, and cut the carrier after GVP2/PUC19 with BamHI, HindIII enzyme and be connected.Enzyme is cut and is shown that obtained sequence is correct sequence with sequencing result.
The structure of embodiment 7 manual transcription factor expression vector
Cut with NheI, HindIII enzyme, reclaim the fragment GVP4 of 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.
Foreign gene high expression in mammalian cell of embodiment 8pATEW mediation
Respectively the foreign gene of coding EGFP, tPA, proUK and Enbrel is connected into High Expression Vectors in Mammalian Cells pATEW by multiple clone site, mammalian cell expression vector pATEW/EGFP, pATEW/tPA, pATEW/proUK and pATEW/Enbrel of construction expression EGFP, tPA, proUK and Enbrel respectively, the structure of each expression vector as shown in Figure 3.
Embodiment 9 efficient expression vector cotransfection mammalian cell
Respectively by pATEW/EGFP, pATEW/tPA, pATEW/proUK and pATEW/Enbrel and manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4 by etc. mole to mix, with transfection reagent Lepofectamine2000 cotransfection Chinese hamster ovary celI and HEK293 cell.
The screening of the exogenous gene high-efficient expressed cell of embodiment 10 and clone
Through the transfection positive CHO cells 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 mediates the expression efficiency of EGFP in Chinese hamster ovary celI and the expression efficiency of commercialization carrier pcDNA3.1 (+) mediation EGFP in Chinese hamster ovary celI improves about 29.3 times, as shown in Figure 5.Or by the clone of limiting dilution assay clone in conjunction with the mensuration of external source target protein and cell counting determination foreign gene expression levels, screening efficiently expressing exogenous gene.Wherein, the mensuration of tPA and proUK adopts external scleroproein agar plate solusphere method; The detection of Enbrel adopts ELISA method.CedexAS20 cell density and vigor automatic analysis system (Innovatis, Germany) is adopted to carry out viable count.Through colony screening, to obtain proUK stably express level be respectively 20-25pg/cell/day, tPA stably express level be 25-30pg/cell/day and Enbrel stably express level is the clone of 50-60pg/cell/day.SDS-PAGE analyzes the Supernatant samples that serum-free feeding culture expresses EnbrelCHO cell, presents obvious Enbrel colored band, as shown in Figure 6 in the position identical with commercialization Enbrel product.
Claims (4)
1. a mediate foreign gene carrier of expressing at mammal cell with high efficient, is characterized in that: described carrier comprises people β-globinMAR, hEF-1 α gene transcription regulation element, manual transcription factor binding site, manual transcription factor GVP4 and posttranscriptional regulatory element WPRE;
The sequence of described people β-globinMAR is as shown in SEQIDNo.1;
Described hEF-1 α gene transcription regulation element comprises nucleotide sequence that the hEF-1 α 5 ' as shown in SEQIDNo.2 holds and the nucleotide sequence that the hEF-1 α 3 ' shown in SEQIDNo.3 holds;
The sequence of described manual transcription factor binding site is as shown in SEQIDNo.4;
The sequence of described posttranscriptional regulatory element WPRE is as shown in SEQIDNo.5;
The nucleotide sequence of described manual transcription factor GVP4 is as shown in SEQIDNo.6;
Described people β-globinMAR, hEF-1 α gene transcription regulation element, posttranscriptional regulatory element WPRE and manual transcription factor binding site are combined into exogenous gene high-efficient expressed carrier pATEW;
Described manual transcription factor GVP4 constructs manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4;
The nucleotide sequence that the hEF-1 α 5 ' of described hEF-1 α gene transcription regulation element holds is positioned at the upstream of the foreign gene of mediation, and the nucleotide sequence that hEF-1 α 3 ' holds 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 that hEF-1 α 5 ' holds;
Described people β-globinMAR is positioned at the upstream of manual transcription factor binding site;
Described posttranscriptional regulatory element WPRE is positioned at the upstream of the nucleotide sequence that hEF-1 α 3 ' holds;
The construction process of described carrier is as follows:
(1) with pcDNA3.1 (+) for carrier framework, the CMV promoter of pcDNA3.1 (+) is replaced with hEF-1 α gene transcription regulation element, the manual transcription factor binding site sequence containing 10 GaL4 binding sites is inserted in the upstream of the nucleotide sequence held at hEF-1 α 5 ', people β-globinMAR is inserted in the upstream of manual transcription factor binding site sequence, at the upstream insertion posttranscriptional regulatory element WPRE of the nucleotide sequence that hEF-1 α 3 ' holds, be built into exogenous gene high-efficient expressed carrier pATEW;
(2) with pcDNA3.1 (+) for carrier framework, the neomycin resistance gene Neo (R) of pcDNA3.1 (+) is replaced with hygromycin gene Hygro (R), insert the nucleotide sequence of 4 tandem repetitive sequences composition manual transcription factor GVP4 of 12 peptides (DALDDFDLDMLG) in 1-147 amino acids and hsv transcription activator VP16 in coding yeast transcription factor GAL4 in the downstream of CMV promoter, build manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4.
2. the mediate foreign gene according to claim 1 construction process of carrier of expressing at mammal cell with high efficient, is characterized in that: described method comprises the steps:
(1) with pcDNA3.1 (+) for carrier framework, the CMV promoter of pcDNA3.1 (+) is replaced with hEF-1 α gene transcription regulation element, the manual transcription factor binding site sequence containing 10 GaL4 binding sites is inserted in the upstream of the nucleotide sequence held at hEF-1 α 5 ', people β-globinMAR is inserted in the upstream of manual transcription factor binding site sequence, at the upstream insertion posttranscriptional regulatory element WPRE of the nucleotide sequence that hEF-1 α 3 ' holds, be built into exogenous gene high-efficient expressed carrier pATEW;
(2) with pcDNA3.1 (+) for carrier framework, the neomycin resistance gene Neo (R) of pcDNA3.1 (+) is replaced with hygromycin gene Hygro (R), insert the nucleotide sequence of 4 tandem repetitive sequences composition manual transcription factor GVP4 of 12 peptides (DALDDFDLDMLG) in 1-147 amino acids and hsv transcription activator VP16 in coding yeast transcription factor GAL4 in the downstream of CMV promoter, build manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4.
3. the application of expression vector according to claim 1 in mediation encoding antibody, Fc fusion rotein, cytokine, thrombin and the expression of somatomedin foreign gene at mammalian cell.
4. utilize carrier described in claim 1 in a mammal cell with high efficient expression method, it is characterized in that, step is as follows:
(1) respectively the cDNA of coding enhanced green fluorescence protein EGFP, tPA, uPA proUK and Enbrel is inserted into the downstream of the nucleotide sequence that hEF-1 α 5 ' holds in described pATEW, is built into carrier pATEW/EGFP, pATEW/tPA, pATEW/proUK and pATEW/Enbrel of mediating different exogenous gene expression;
(2) respectively pATEW/EGFP, pATEW/tPA, pATEW/proUK and pATEW/Enbrel and manual transcription factor expression vector pcDNA3.1 (+)/Hy/GVP4 are pressed equimolar ratio cotransfection Chinese hamster ovary celI or HEK293 cell;
(3) clone of efficiently expressing exogenous gene is screened.
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| CN103305540B (en) * | 2012-03-14 | 2015-03-25 | 齐鲁制药有限公司 | Plasmid for determination of biological activity and preparation method thereof |
| CN102703503B (en) * | 2012-06-01 | 2013-08-28 | 新乡医学院 | Human and mammal cell attachment expression vector and application thereof |
| 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 |
| CN105039411B (en) * | 2015-08-27 | 2019-01-18 | 新乡医学院 | Attached type slow virus carrier, preparation method and application |
| CN114891829B (en) * | 2022-05-24 | 2023-09-26 | 新乡医学院 | Liver-specific episomal expression vector, gene therapy vector and application thereof |
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