CN107058387B - A kind of two-cistron expression vector of suitable HEK293 cell and preparation method thereof, expression system, application - Google Patents
A kind of two-cistron expression vector of suitable HEK293 cell and preparation method thereof, expression system, application Download PDFInfo
- Publication number
- CN107058387B CN107058387B CN201710240457.3A CN201710240457A CN107058387B CN 107058387 B CN107058387 B CN 107058387B CN 201710240457 A CN201710240457 A CN 201710240457A CN 107058387 B CN107058387 B CN 107058387B
- Authority
- CN
- China
- Prior art keywords
- sequence
- pires
- expression
- cell
- mar
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron
- C12N2830/205—Vector systems having a special element relevant for transcription transcription of more than one cistron bidirectional
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to two-cistron expression vectors of a kind of suitable HEK293 cell and preparation method thereof, expression system, application, belong to gene engineering technology field.The two-cistron expression vector of suitable HEK293 cell of the invention includes following element: the first matrix attachment region sequence-promoter-target gene-internal ribosome entry site sequence-matrix attachment region riddled basins-Poly A-the second sequence.The carrier can significantly improve HEK293 cell transgene expression using the MAR sequence of optimization, overcome transgene silencing, realize efficient, long-term expression of the transgenosis in host cell.Expression while can realizing target gene and selection markers simultaneously can reduce since there are caused false positive cell clones for different expression cassettes, improve positive cell clone screening rate, and it is unbalanced to overcome the problems, such as that conventional carriers target gene is expressed with riddled basins.
Description
Technical field
A kind of two-cistron expression vector of suitable HEK293 cell and preparation method thereof, expression system, application, belong to base
Because of field of engineering technology.
Background technique
The 1970s technique for gene engineering development, make to utilize animal viscera, tissue or human blood to extract originally
Pharmaceutical grade protein is changed into be produced using genetic engineering.Pharmaceutical protein includes monoclonal antibody, polypeptide, albumen, has become doctor
The pith of medicine industry, and the key areas that the following medicine development will be become.
The expression system of recombinant pharmaceutical proteins mainly includes E.coli, yeast and non-humanization mammal cell line.
E.coli is suitble to express that molecular weight is smaller, the relatively simple protein of structure, yeast expression system be suitble to express molecular weight it is larger,
Structure is more complex, less glycosylated protein.Structure is complicated or glycosylation modified extremely important for the activity of protein, non-
Humanization mammalian cell is due to having the post translational processing modification (post-translational for being similar to human cell
Modifications, PTMs), therefore, non-humanization mammalian cell expression system is that current drug recombinant pharmaceutical proteins are raw
The Important Platform of production.Common non-humanization mammalian cell is specifically included that such as Chinese hamster ovary (Chinese
Hamster ovary, CHO), newborn Hamster Embryonic Kidney cell (Baby Hamster Kidney, BHK), NSO mouse thymus oncocyte,
SP2/0 myeloma cell, NSO murine myeloma cell.Although non-humanization mammal cell expresses recombinant pharmaceutical proteins
Have safety and validity, but since PTMs mode is different, especially glycosylation modified there are difference with human archeocyte.Such as
Murine cells lack the glycosylated mechanism of human archeocyte, and expression albumen is easy that human body is made to generate immunity, lead to external weight
Group pharmaceutical protein is removed by the circulatory system.HEK293 cell origin was utilized by Graham in 1977 earliest in human embryonic kidney cells
It transfects Adenovirus Type 5 DNA to obtain, is applied to the production of recombinant pharmaceutical proteins and viral vectors in recent years.HEK293 can be
SFM suspension growth, easily transfection have the advantages of production recombinant pharmaceutical proteins.HEK293 cell is tamed and is transformed,
Cell line rapid, that transfection efficiency is high, expression is high can be grown in SFM by cultivating.Have 5 kinds so far by HEK293 cell line
The pharmaceutical protein of production obtains U.S. FDA or EMA approval production.
That there is also destination protein expressions is low for HEK293 cell expression system, the high and stable yields cell screening period is long, thin
The defects of born of the same parents' toxigenic capacity is high, this seriously restricts the production of recombinant protein medicine.And recombinant protein is influenced in HEK293 cell
The factor of middle expression is numerous, and expression vector is exactly one of key factor.In addition, high level is held in cell expression system
The continuous cell strain for stablizing expression is often difficult to obtain, this is because the random integration after transgenosis transfection cell, makes some turn base
It is reduced because silencing or expression occurs, it is therefore desirable to the clone of stability and high efficiency expression is isolated from a large amount of cell clones
Cell strain, this also brings very big puzzlement to genetic engineering industrialization production.
Matrix attachment region sequence (matrix attachment region, MAR) is remained stuck to after limiting enzymic digestion
DNA sequence dna on paralinin, length are 300~3000bp, rich in AT base-pair (> 60%) and several short " shared sequences
Column ", as A-box (AATAAAYAAA), T-box (TTWTWTTWTT), DNA chain untwist sequence (AATATATTT or AATATT), open up
Flutter isomerase II binding site (GTNWAYATTNATNNR) etc..Studies have shown that MAR sequence can improve HEK293 cell attachment
Carrier (episomal vectors) transgene expression level, while reducing transformant transgene expression level difference
(Modified S/MAR episomal vectors for stably expressing fluorescent protein-
tagged transgenes with small cell-to-cell fluctuations.Anal Biochem.2013).Together
When research it has also been found that the MAR of wild boar can improve HEK293 cell transgene expression level (Sus scrofa matrix
attachment region enhances expression of the PiggyBac system transfected into
HEK293T cells.Biotechnol Lett.2016).Existing research proves that MAR is improved on transgene expression and carrier
Other controlling elements (regulatory elements) are related, as MAR is combined with different promoters to the table for improving recombinant protein
Up to horizontal not first-class (Impact of Using Different Promoters and Matrix Attachment
Regions on Recombinant Protein Expression Level and Stability in Stably
Transfected CHO Cells.Mol Biotechnol.2014)。
The Chinese invention patent of Publication No. CN102994536A discloses a kind of bicistronic mRNA and shifts body for expressing gene
And preparation method thereof, disclosing and playing 3 ' ends from 5 ' ends only successively includes ox matrix attachment region MAR, artificial constructed combination
Promoter CAG, inhibit protein gene FSTN, internal ribosome entry site IRES, green fluorescent protein AcGFP gene,
The expression vector of Rabbit globin polyA signaling zone, but its MAR sequence derives from ox, is not suitable for people source
HEK293 cell line, and the expression vector does not include selection markers, not can be carried out the screening of stable transformed cells strain.It is open
Number a kind of two-cistron expression vector is disclosed for the Chinese invention patent of CN106520832A, but due to different MAR members
Part combination also has different role (Matrix attachment region combinations to the expression of transgenosis
increase transgene expression in transfected Chinese hamster ovary cells.Sci
Rep.2017), related (the Cell compatibility of an of host cell that the skeleton structure of carrier is transfected with it
eposimal vector mediated by the characteristic motifs of matrix attachment
Regions.Curr Gene Ther, 2016), the carrier of expressing cho cell is suitble to not necessarily to be suitble to HEK293 cell
(Evaluating post-transcriptional regulatory elements for enhancing transient
Gene expression levels in CHO K1and HEK293cells.Protein Expr Purif.2010), therefore
It researches and develops and is suitble to the expression vector of HEK293 cell significant.
Summary of the invention
The purpose of the present invention is to provide a kind of two-cistron expression vectors of suitable HEK293 cell.
The present invention also provides the preparation methods of above-mentioned two-cistron expression vector.
The present invention also provides the expression systems of above-mentioned two-cistron expression vector.
The present invention also provides the applications of above-mentioned two-cistron expression vector.
To achieve the goals above, the technical scheme adopted by the invention is that:
A kind of two-cistron expression vector of suitable HEK293 cell includes following element: the first matrix attachment region sequence
Column-promoter-target gene-internal ribosome entry site sequence-riddled basins-Poly A-the second paralinin combines
Region sequence.
The first matrix attachment region sequence behaviour X-29 MAR sequence, human beta-globin MAR sequence, chicken egg lysozyme
MAR sequence, people's HPRT MAR sequence or MAR 1-68 sequence；The second matrix attachment region sequence behaviour X-29 MAR sequence
Column, human beta-globin MAR sequence, chicken egg lysozyme MAR sequence, people's HPRT MAR sequence or MAR 1-68 sequence.
First matrix attachment region sequence and the second matrix attachment region sequence behaviour X-29 MAR sequence, people β-pearl
Any two kinds in albumen MAR sequence, chicken egg lysozyme MAR sequence, people's HPRT MAR sequence or MAR 1-68 sequence.
Preferably, first matrix attachment region sequence and the second matrix attachment region sequence are the paralinin in people source
In conjunction with region sequence.
Preferably, the first matrix attachment region sequence behaviour X-29 MAR sequence, GenBank:EF694970.1, the
1~3337 bit base.
Preferably, it is GenBank:L22754.1 that second matrix attachment region sequence, which is human beta-globin MAR sequence,
840th~2998 bit base.
The chicken egg lysozyme MAR sequence is GenBank:X98408, the 1st~1668 bit base；
The people HPRT MAR sequence is GenBank:X07690, the 1st~580 bit base.
The MAR 1-68 is GenBank:EF694965.1, the 1st~3614 bit base.
The promoter is one of SV40 promoter, CMV promoter, EF-1 α promoter.
Preferably, the promoter is CMV promoter, and nucleotide sequence is as shown in SEQ ID NO.1.
The riddled basins are neomycin phosphotransferase gene or blasticidin resistance gene.
Preferably, the riddled basins are that NPT resistance weakens gene, nucleotide sequence such as SEQ ID NO.2 institute
Show.
The target gene is drug recombinant protein gene.
Preferably, the target gene is erythropoietin gene.
As one embodiment of the present invention, the two-cistron expression vector is to set out with pIRES-Neo carrier
Vector construction.
The carrier that sets out is the starting vector that can be transformed according to demand.
The HEK293 cell is HEK293-H, HEK293-F or HEK293-T cell.
The construction method of above-mentioned two-cistron expression vector: include the following steps:
1) riddled basins are connected on pIRES-Neo carrier, obtain pIRES-H1 carrier；
2) the first matrix attachment region sequence and second are inserted into before the promoter of pIRES-H1 carrier and respectively after polyA
Matrix attachment region sequence is to get pIRES-H2 carrier.
Riddled basins are connected to the concrete operations of pIRES-Neo carrier described in step 1) are as follows: using XmaI and
Sequence and pIRES-neo carrier shown in XbaI enzyme double digestion SEQ ID NO.3, recycle endonuclease bamhi, and connection is converted to large intestine bar
Identification, obtains pIRES-H1 carrier after in bacterium.
Described in step 2) matrix attachment region sequence is inserted into before the promoter of pIRES-H1 carrier and respectively after polyA
Concrete operations are as follows: using NruI and MluI double digestion synthesis people X-29 MAR sequence and pIRES-H1 Plasmid DNA, recycle digestion
Segment, connection are identified after converting into Escherichia coli, obtain pIRES-H1M carrier；Using XhoI and BstZ17I double digestion β-pearl
Albumen MAR sequence and pIRES-H1M Plasmid DNA, connection are identified after converting into Escherichia coli, obtain pIRES-H2 carrier.
The HEK293 expression system that above-mentioned two-cistron expression vector constructs.
The construction method of above-mentioned HEK293 expression system: including: to transfect the two-cistron expression vector to enter
In HEK293 cell to get.
The transfection is calcium phosphate method, electric robin, lipofection transfection.
Preferably, the transfection is electric robin transfection.
Above-mentioned two-cistron expression vector is preparing the application in pharmaceutical protein.
The two-cistron expression vector that the present invention provides a kind of suitable HEK293 cell includes matrix attachment region sequence, interior
Ribosomal entry site sequence and riddled basins can structures by being inserted into target gene between promoter and IRES sequence
At target gene-IRES sequence-sequentially connected bicistronic mRNA sequence of riddled basins.The structure of expression vector are as follows: MAR-
Promoter-target gene-IRES sequence-riddled basins-PolyA-MAR.Carrier of the invention contains suitable HEK cell line
The MAR sequence of expression, and the selection markers containing reduction, the carrier can be significantly improved using the MAR sequence of optimization
HEK293 cell transgene expression overcomes transgene silencing, realizes efficient, long-term expression of the transgenosis in host cell.Together
When expression while can realize target gene and selection markers, can reduce since there are caused false positive cells for different expression cassettes
Clone improves positive cell clone screening rate, and it is unbalanced to overcome conventional carriers target gene to express with riddled basins
Problem.
Detailed description of the invention
Fig. 1 is pIRES-Neo carrier structure schematic diagram in embodiment 1；
Fig. 2 is the pIRES-H1 carrier structure schematic diagram in embodiment 1；
Fig. 3 is the pIRES-H2 carrier structure schematic diagram in embodiment 1；
Fig. 4 is expression statistical chart of the EPO in HEK293 cell monoclonal in embodiment 2；
Fig. 5 compares figure in the Average expression level of different carriers stable screening for EPO.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Escherichia coli (Escherichia coli) JM109, pIRES- used in embodiment and test example
Neo plasmid vector, cell line reagent, toolenzyme etc. are commercial goods.Restriction endonuclease and NEBuffer are purchased from U.S. New
England Biolabs LTD (NEB), pIRES-neo plasmid vector are purchased from Clontech biotech firm.
Embodiment 1
It is suitble to the building of the two-cistron expression vector of HEK293 cell, the specific steps are as follows:
1, the building of pIRES-H1 carrier
The purpose of this step is to replace with the riddled basins on pIRES-neo carrier (its structure is as shown in Figure 1)
NPT resistance weakens gene.
1) artificial synthesized NPT resistance weakens gene
It designs and artificial synthesized NPT resistance weakens gene (as shown in SEQ ID NO.3), specifically transfer to general biological gene
(Anhui) Co., Ltd completes.
For convenient for cloning and guaranteeing sequence integrity, when synthesis NPT resistance weakens gene, 5 ' ends introduce AAC CCCGGG
ATAATTCCTGCAGCCAAT sequence, wherein AAC is protection base, and CCCGGG is XmaI restriction enzyme site,
ATAATTCCTGCAGCCAAT be carrier pIRES-Neo on partial sequence (GenBank:U89673.1, the 1892nd~1909
Base)；3 ' ends introduce GGGGATCAATTC TCTAGA GCT sequence, and wherein GCT is protection base, and TCTAGA is XbaI enzyme cutting
Site, GGGGATCAATTC be carrier pIRES-Neo on partial sequence (GenBank:U89673.1, the 2714th~2725
Base).
2) pIRES-H1 carrier is constructed
Gene is weakened with the NPT resistance that XmaI/XbaI double digestion synthesizes, while with XmaI/XbaI double digestion pIRES-neo
Plasmid DNA, then with NEB companyTM25 DEG C of connection 5min of connection kit.Connection product is added to E.coli JM109 bacterial strain
It is converted in competent cell suspension, takes 150 μ L transformed bacteria solutions to be inoculated on the LB plate containing ampicillin, 37 DEG C were cultivated
Night, picking single colonie squamous subculture.It extracts recombinant plasmid and carries out double digestion (XmaI/XbaI) verifying, take digestion verification correct
Plasmid carry out sequence verification, construct correct plasmid and be named as pIRES-H1, structural schematic diagram is as shown in Figure 2.
2, the building of pIRES-H2 carrier
The purpose of this step is to be inserted into people X-29 MAR sequence and poly in the upstream of the promoter CMV of pIRES-H1 carrier
Beta-globin MAR sequence is inserted into the downstream of A.
1) people X-29 MAR sequence is synthesized
According to people X-29 MAR sequence (GenBank:EF694970.1, the 1st~3337 bit base), general life is specifically transferred to
Object gene (Anhui) Co., Ltd completes.For convenient for clone, when synthesizing people X-29 MAR sequence, the end 5' introduces GTCTCGCGA sequence
Column, wherein GTC is protection base, and TCGCGA is NruI restriction enzyme site.3 ' ends introduce AGCACGCGT, and wherein AGC is as digestion
Base is protected, ACGCGT is MluI restriction enzyme site.
2) expression vector of promoter CMV upstream X-29 containing people MAR sequence is constructed
The people's X-29 MAR sequence synthesized with NruI/MluI double digestion, while with NruI/MluI double digestion pIRES-H1 matter
Grain DNA.Agarose gel electrophoresis identifies digestion as a result, gel recycles the people's X-29 MAR sequence fragment and pIRES-H1 after digestion
Shape material grain DNA.
The double digestion system of people's X-29 MAR sequence are as follows: 10 μ L of people X-29 MAR sequence (1 μ g/ μ L), 10 × NEBuffer
3.1 3 μ L, NruI/MluI (10U/ μ L) each 1.0 μ L, supplies water to 30 μ L；Digestion condition are as follows: 37 DEG C, digestion 3min.
The double digestion system of pIRES-H1 plasmid are as follows: 5 μ L of pIRES-H1 plasmid (1 μ g/ μ L), 10 × NEBuffer 3.1 2
μ L, NruI/MluI (10U/ μ L) each 0.5 μ L, supplies water to 20 μ L；Digestion condition are as follows: 37 DEG C, digestion 3min.
People's X-29 MAR sequence fragment and pIRES-H1 shape material grain DNA (molar ratio 5:1) after taking digestion, use NEB
CompanyTMConnection kit, 25 DEG C of connection 5min.Connection product is added to E.coli JM109 bacterial strain competent cell suspension
Middle conversion takes 150 μ L transformed bacteria solutions to be inoculated on the LB plate containing ampicillin, 37 DEG C of overnight incubations, picking single colonie
Squamous subculture.It extracts recombinant plasmid and carries out double digestion (NruI/MluI) verifying, the correct plasmid of digestion verification is taken to be sequenced
Verifying, constructs correct plasmid and is named as pIRES-H1M.
3) PCR amplification beta-globin MAR sequence
According to beta-globin MAR sequence (GenBank:L22754.1, the 840th~2998) design primer P1, P2,.?
5 ' ends, the 3 ' ends of primer P1, P2 introduce 5 ' ends respectively and introduce XhoI, BstZ17I restriction enzyme site respectively, primer sequence it is following (under
It is marked as restriction enzyme site):
Primer P1:5 '-GTCCTCGAGAATATATCTCCTGATAAAATGTCTA-3 ' (as shown in SEQ ID NO.4)；
Primer P2:5 '-GTCGTATACGGATCCTCCCATTTCGGCCTCCTG-3 ' (as shown in SEQ ID NO.5).
Human peripheral genomic DNA is extracted as template, carries out PCR amplification, amplification reaction system with primer P1/P2 respectively
It see the table below 1.
1 pcr amplification reaction system of table
| PCR reaction system | Reagent concentration | Volume (μ L) | Final concentration |
| ddH2O | / | 17 | / |
| P1/P2 | 10μmol/L | Each 1.0 | 0.4μmol/L |
| 10×PCR buffer | / | 2.5 | 1× |
| dNTP | 25μmol/L | 2.0 | 200μmol/L |
| Template DNA | 100ng/μL | 1.0 | 4.0ng/μL |
| Taq enzyme | 5U/μL | 0.5 | 0.1U/μL |
PCR amplification program are as follows: 95 DEG C of 3min, 94 DEG C of 40s, 58 DEG C of 30s, 72 DEG C of 40s, each annealing temperature 4 circulations, most
55 DEG C afterwards, 30 circulations, 72 DEG C of 3min.
Agarose gel electrophoresis recycles amplified production, send biotech firm's sequence verification.The result shows that the DNA piece amplified
The beta-globin MAR sequence that Duan Junyu GenBank is logged in is consistent.
4) expression for constructing promoter CMV upstream X-29 containing people MAR and poly A downstream MAR containing beta-globin sequence carries
Body
The beta-globin MAR sequence amplified with XhoI/BstZ17I double digestion P1/P2, at the same it is bis- with XhoI/BstZ17I
Digestion pIRES-H1M Plasmid DNA.Agarose gel electrophoresis identifies digestion as a result, gel recycles the beta-globin MAR sequence after digestion
Column-slice section and pIRES-H1M shape material grain DNA.Use NEB companyTMConnection kit, 25 DEG C of connection 5min.By connection product
It is added in E.coli JM109 bacterial strain competent cell suspension and converts, 150 μ L transformed bacteria solutions is taken to be inoculated into containing ammonia benzyl mould
On the LB plate of element, 37 DEG C of overnight incubations, picking single colonie squamous subculture.It extracts recombinant plasmid and carries out double digestion (XhoI/
BstZ17I it) verifies, takes the correct plasmid of digestion verification to carry out sequence verification, construct correct plasmid and be named as pIRES-H2,
Structural schematic diagram is as shown in Figure 3.
Embodiment 2
The building of eukaryotic cell expression system, the specific steps are as follows:
1, building contains the two-cistron expression vector of hematopoietin (EPO) foreign gene
1) EPO sequence is synthesized
According to the EPO sequence (GenBank:JN849371.1, the 1st~582 bit base) that NCBI is announced, artificial synthesized EPO
Sequence (5 ' ends and 3 ' ends introduce EcoRI, BamHI restriction enzyme site respectively), specifically transfers to general biological gene (Anhui) limited public affairs
Department completes.
2) two-cistron expression vector of the sequence containing EPO is constructed
With the EOP sequence that EcoRI/BamHI double digestion primer is artificial synthesized, while with EcoRI/BamHI double digestion
PIRES-neo and pIRES-H2 Plasmid DNA.Agarose gel electrophoresis identifies digestion as a result, gel recycles the EPO sequence after digestion
Segment and pIRES-neo, pIRES-H2 shape material grain DNA.
The double digestion system of EPO sequence are as follows: 10 μ L of EPO sequence fragment (1 μ g/ μ L), 10 × NEBuffer, 2.1 3 μ L,
EcoRI/BamHI enzyme (10U/ μ L) each 1.0 μ L, supplies water to 30 μ L；Digestion condition are as follows: 37 DEG C, digestion 3min.
The double digestion system of pIRES-H2 plasmid are as follows: 5 μ L of pIRES-H2 plasmid (1 μ g/ μ L), 10 × NEBuffer 2.1 2
μ L, EcoRI/BamHI (10U/ μ L) each 0.5 μ L, supplies water to 20 μ L；Digestion condition are as follows: 37 DEG C, digestion 3min.
EPO sequence fragment and pIRES-H2 shape material grain DNA (molar ratio 5:1) after taking digestion, use NEB companyTM's
Connect kit, 25 DEG C of connection 5min.Connection product is added in E.coli JM109 bacterial strain competent cell suspension and is converted,
150 μ L transformed bacteria solutions are taken to be inoculated on the LB plate containing ampicillin, 37 DEG C of overnight incubations, picking single colonie is after being commissioned to train
It supports.It extracts recombinant plasmid and carries out double digestion (EcoRI/BamHI) verifying, take the correct plasmid of digestion verification to carry out sequencing and test
Card, constructs correct plasmid and is named as pIRES-H2-EPO.
2, eukaryotic cell expression system is constructed
Select the good HEK293 cell inoculation to 6 well culture plates of growth conditions on, to plating density reach about 80% into
Row transfection.Specific steps are as follows: 10 μ L lipofectamine 2000+240 μ L serum-free FreeStyleTM 293
Expression Medium stands 5min in 37 DEG C of incubators, by serum free medium and 250 μ L (5 μ g) expression vector
PIRES-H2-EPO is mixed, and stands 20min in 37 DEG C of incubators；The cell on 6 well culture plates is cleaned three times with PBS simultaneously, is added
Enter 2mL serum-free cell culture medium；Then by the mixed liquor of liposome and the Plasmid DNA containing pIRES-H2-EPO light inlet hole dropwise
In, jiggling culture plate as early as possible makes its mixing；It is put into 5%CO2In cell incubator.600 μ g/mL are added in cell after 48h
G418 drug, every 48h changes fresh complete serum free medium into, the cell mortality since the 5th day.Screening is after two weeks
G418 concentration is adjusted to 300 μ g/mL of concentration is maintained to continue to cultivate.After the completion of drug screening, limiting dilution assay is carried out to cell
Monoclonalization operation.About fortnight monoclonal cell takes cell conditioned medium to do ELISA detection after Kong Zhongchang to 80%, one day,
Testing result is shown in Fig. 4.As shown in Figure 4, in 10 clones selected, the yield of 1# and 4# clone are best, respectively 256.2mg/L
And 248.3mg/L.
Embodiment 3
It is suitble to the building of the two-cistron expression vector pIRES-Neo-EPO of HEK293 cell.
Referring to the pIRES-Neo-EPO expression vector of method building foreign gene containing EPO in embodiment 2, with pIRES-
The difference of H2-EPO are as follows:
1) selection markers are different；PIRES-Neo-EPO selection markers be carrier on 1906~2709 (GenBank:
U89673.1, the 1906th~2709 bit base)；PIRES-H2-EPO selection markers sequence is as shown in SEQ ID NO.2；
2) pIRES-Neo-EPO does not contain the MAR sequence at both ends, pIRES-H2-EPO X- containing someone before CMV promoter
29 MAR sequences contain beta-globin MAR sequence in the downstream of poly A.
PIRES-Neo-EPO eukaryotic cell expression system is constructed, construction method is the same as embodiment 2.
Embodiment 4
The building of the two-cistron expression vector of suitable HEK293 cell in the present embodiment, includes the following steps:
It is the carrier that sets out with pIRES-Neo carrier, people X-29 MAR sequence is inserted into the upstream of promoter CMV, in poly
People HPRT MAR sequence is inserted into the downstream of A, and insertion point is the same as embodiment 1.
Test example
By pIRES-Neo-EPO expression vector, pIRES-H2-EPO expression vector and Publication No. CN 106520832A
Chinese invention patent in pIRES-C3-EPO expression vector using identical operation transfect into HEK293 cell, acquisition number
Approximate monoclonal cell strain is measured, ELISA detection is carried out to positive monoclonal group, and compare the expression quantity of clone strain, respectively sieved
Choosing obtains 10 stable cell lines, analyzes the EPO expression quantity of 10 plants of cells.
Result is analyzed as shown in figure 5, the results show that the EPO expression using expression system of the present invention is apparently higher than biography
The expression system of system, EPO expression quantity the average out to 196.98mg/L, pIRES-C3-EPO of stable screening express EPO gene expression
Amount reaches 62.65mg/L, and the EPO expression quantity of conventional expression system pIRES-Neo-EPO is only 39.45mg/L, of the invention
Expression system improves 3.14 times with respect to pIRES-C3-EPO, and opposite pIRES-Neo-EPO improves 4.99 times (P < 0.01), and discloses
Though the pIRES-C3-EPO of number Chinese invention patent for being 106520832 A of CN increases with control, significantly (only
Improve 1.59 times).
SEQUENCE LISTING
<110>Xinxiang College of Medical Science
<120>a kind of two-cistron expression vector of suitable HEK293 cell and preparation method thereof, expression system, application
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 599
<212> DNA
<221>CMV promoter
<222> (1)..(599)
<400> 1
agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca 60
ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 120
ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 180
acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac 240
ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 300
aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 360
tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 420
gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 480
gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 540
ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctct 599
<210> 2
<211> 795
<212> DNA
<221>NPT resistance weakens gene
<222> (1)..(795)
<400> 2
atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 540
ggcgatgatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
aacgagttct tctga 795
<210> 3
<211> 843
<212> DNA
<221>synthesis NPT resistance weakens gene
<222> (1)..(843)
<400> 3
aaccccggga taattcctgc agccaatatg attgaacaag atggattgca cgcaggttct 60
ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc 120
tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc 180
gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc 240
acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg 300
ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag 360
aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc 420
ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt 480
cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc 540
gccaggctca aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc 600
tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg 660
ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag 720
cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg 780
cagcgcatcg ccttctatcg ccttcttaac gagttcttct gaggggatca attctctaga 840
gct 843
<210> 4
<211> 34
<212> DNA
<213>artificial sequence
<221>primer P1
<222> (1)..(34)
<400> 4
gtcctcgaga atatatctcc tgataaaatg tcta 34
<210> 5
<211> 33
<212> DNA
<213>artificial sequence
<221>primer P2
<222> (1)..(33)
<400> 5
gtcgtatacg gatcctccca tttcggcctc ctg 33
Claims (4)
1.HEK293 expression system, it is characterised in that: the HEK293 expression system is constructed to obtain by two-cistron expression vector；
By the two-cistron expression vector transfection enter HEK293 cell in get；The two-cistron expression vector includes as follows
Element: people X-29 MAR sequence-CMV promoter-target gene-internal ribosome entry site sequence-riddled basins-
Poly A-human beta-globin MAR sequence；The people X-29 MAR sequence be GenBank:EF694970.1, the 1st~3337
Base；The human beta-globin MAR sequence is GenBank:L22754.1, the 840th~2998 bit base；The selection markers base
Because NPT resistance weakens gene, nucleotide sequence is as shown in SEQ ID NO .2.
2. HEK293 expression system according to claim 1, it is characterised in that: the two-cistron expression vector with
PIRES-Neo carrier is the vector construction that sets out.
3. HEK293 expression system as described in claim 1, it is characterised in that: the preparation side of the two-cistron expression vector
Method includes the following steps:
1) riddled basins are connected on pIRES-Neo carrier, obtain pIRES-H1 carrier；
2) people's X-29 MAR sequence and human beta-globin MAR are inserted into before the promoter of pIRES-H1 carrier and respectively after polyA
Sequence is to get pIRES-H2 carrier.
4. HEK293 expression system as described in claim 1 is preparing the application in pharmaceutical protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710240457.3A CN107058387B (en) | 2017-04-13 | 2017-04-13 | A kind of two-cistron expression vector of suitable HEK293 cell and preparation method thereof, expression system, application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710240457.3A CN107058387B (en) | 2017-04-13 | 2017-04-13 | A kind of two-cistron expression vector of suitable HEK293 cell and preparation method thereof, expression system, application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107058387A CN107058387A (en) | 2017-08-18 |
| CN107058387B true CN107058387B (en) | 2019-09-06 |
Family
ID=59599458
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710240457.3A Active CN107058387B (en) | 2017-04-13 | 2017-04-13 | A kind of two-cistron expression vector of suitable HEK293 cell and preparation method thereof, expression system, application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107058387B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BR112020007772A2 (en) * | 2017-10-25 | 2020-10-06 | Nouscom Ag | cell line, use of cell line and method to produce infectious viral particles |
| CN110484563B (en) * | 2019-07-25 | 2023-04-07 | 新乡医学院 | Mammal cell combined expression vector, expression system, preparation method and application |
| CN114438124A (en) * | 2022-01-27 | 2022-05-06 | 新乡医学院 | Expression vector for CHO cell protein expression system and screening method of CHO cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994536A (en) * | 2013-01-08 | 2013-03-27 | 内蒙古大学 | Bicistronic mRNA coexpression gene transporter and preparation method thereof |
| WO2014017851A2 (en) * | 2012-07-27 | 2014-01-30 | Hanwha Chemical Corporation | Novel mars and method for producing target protein using the same |
| CN106520832A (en) * | 2016-11-17 | 2017-03-22 | 新乡医学院 | Bicistronic expression vector, expression system, preparation method and application |
-
2017
- 2017-04-13 CN CN201710240457.3A patent/CN107058387B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014017851A2 (en) * | 2012-07-27 | 2014-01-30 | Hanwha Chemical Corporation | Novel mars and method for producing target protein using the same |
| CN102994536A (en) * | 2013-01-08 | 2013-03-27 | 内蒙古大学 | Bicistronic mRNA coexpression gene transporter and preparation method thereof |
| CN106520832A (en) * | 2016-11-17 | 2017-03-22 | 新乡医学院 | Bicistronic expression vector, expression system, preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| Evaluating post-transcriptional regulatory elements for enhancing transient gene expression levels in CHO K1 and HEK293 cells;Mariati et al.;《Protein Expression and Purification》;20090827;第69卷;第9-15页 |
| Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells;Chun-Peng Zhao et al.;《Scientific Reports》;20170220;第7卷;图1、图4、图5 |
| Modified S/MAR episomal vectors for stably expressing fluorescent protein-tagged transgenes with small cell-to-cell fluctuations;Akiko Mizutani et al.;《Analytical Biochemistry》;20130819;第443卷;图1，第116页左栏最后一段 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107058387A (en) | 2017-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106520832B (en) | Bicistronic expression vector, expression system, preparation method and application | |
| CN107058387B (en) | A kind of two-cistron expression vector of suitable HEK293 cell and preparation method thereof, expression system, application | |
| CN107723276B (en) | Construction method and kit for cell strain of stable high-expression target product | |
| KR950704502A (en) | ACTIVATING EXPRESSION OF AN AMPLIFYING ENDOGENOUS GENE BY HOMOLOGOUS RECOMBINATION | |
| CA2214018C (en) | An immortalized mammal hybridoma fusion partner | |
| US9150881B2 (en) | Production of proteins using transposon-based vectors | |
| US20090042299A1 (en) | Vectors and methods for tissue specific synthesis of proteins in eggs of transgenic hens | |
| US9150880B2 (en) | Vectors for production of antibodies | |
| US20080235813A1 (en) | Gene regulation in transgenic animals using a transposon-based vector | |
| CN1533432A (en) | COmpositions and methods for high-level, large-scale production of recombinant proteins | |
| KR102365484B1 (en) | Improved recombinant protein expression using a hybrid chef1 promoter | |
| CN111733183A (en) | Targeting vector for constructing liver injury mouse model, nucleic acid composition and construction method | |
| CN114805500B (en) | Application of African swine fever virus I73R protein as immunosuppressant and construction of immunosuppression site mutant strain | |
| KR100868594B1 (en) | Host Cells Containing Multiple Integrating Vectors | |
| EP1539785B1 (en) | Gene regulation in transgenic animals using a transposon-based vector | |
| CN102329784A (en) | Japanese encephalitis virus like particles as well as preparation method and application thereof | |
| CN109402071B (en) | Recombinant turkey herpesvirus expressing H9N2 subtype avian influenza virus H9 protein | |
| CN106536722A (en) | Method for rapid generation of infectious RNA virus | |
| KR20220080085A (en) | Treatment of chronic granulomatous disease | |
| CN101684476A (en) | Transgenosis construct and application thereof in preparing space-time adjustable liver damage model | |
| US20090083872A1 (en) | Production of Proteins in Eggs | |
| CN106929483A (en) | Express structure and its application of the recombinant herpesvirus of turkeys of F gene of Newcastle disease virus | |
| AU2003286135A1 (en) | High yield heterologous expression cell lines for expression of gene products with human glycosylation pattern | |
| CN101855352A (en) | Mammary gland-specific human erythropoietin expression vector, transgenic animal and method for producing human erythropoietin using same | |
| CN101659966A (en) | Method for constructing universal carrier of I type poultry Marek's virus vaccine strain gene engineering and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201105 Address after: 453700 comprehensive R & D building V (A-F) (2-3) - 1, torch Park, high tech Zone, 1789 Xinfei Avenue, Xinxiang City, Henan Province Patentee after: HENAN PUNUOYI BIOLOGICAL PRODUCT RESEARCH INSTITUTE Co.,Ltd. Patentee after: XINXIANG MEDICAL University Address before: Xinxiang Jinsui Avenue East of 453003 cities in Henan Province Patentee before: XINXIANG MEDICAL University |