CN106987559A - A kind of construction method of recombinant C HOK1 cell lines and its application - Google Patents
A kind of construction method of recombinant C HOK1 cell lines and its application Download PDFInfo
- Publication number
- CN106987559A CN106987559A CN201710172903.1A CN201710172903A CN106987559A CN 106987559 A CN106987559 A CN 106987559A CN 201710172903 A CN201710172903 A CN 201710172903A CN 106987559 A CN106987559 A CN 106987559A
- Authority
- CN
- China
- Prior art keywords
- ebna1t
- cell
- recombinant protein
- transfection
- expression vector
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- C12N2510/00—Genetically modified 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
- C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- 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
- C12N2820/00—Vectors comprising a special origin of replication system
- C12N2820/60—Vectors comprising a special origin of replication system from viruses
Abstract
The present invention discloses a kind of construction method for the recombinant C HOK1 cell lines that can stablize expression truncated-type EBNA1 albumen, comprises the following steps：(1) structure of EBNA1t expression vectors, will be such as SEQ ID NO:Sequence shown in 1 is cloned into pWX1.0 and pWX2.0 carriers；(2) transfection is screened with cell mass；(3) limiting dilution assay is cloned, and selects monoclonal cell strain.It is used for the application of recombinant protein transient expression invention additionally discloses the recombinant C HOK1 EBNA1t cell lines that the above method is obtained, comprises the following steps：(1) recombinant protein transient expression vector is built；(2) passage culture is carried out before transfecting；(3) transfection and cell culture process, the CHOK1 EBNA1t cell lines obtained using PEI recombinant protein transient expression vector transfection procedure (2) Secondary Cultures for obtaining step (1)；(4) expression quantity of recombinant protein is assessed.The recombinant C HOK1 EBNA1t cell lines of the present invention are used for recombinant protein transient expression, and not only expression quantity is high, generally 100 300mg/L, and technique is easy, are not only suitable for large volume production, are also applied for small size high flux screening.
Description
Technical field
The present invention relates to bio-pharmaceuticals and biological technical field, expression truncated-type EBNA1 can be stablized more particularly, to one kind
The construction method of the recombinant C HOK1 cell lines of albumen and its application in recombinant protein transient expression.
Background technology
With the development of biotech drug, milligram can be quickly produced to the recombinant protein Transient Expression System of gram rank
It is widely used in new medicament screen and preclinical study.Chinese hamster ovary celI is the most frequently used place in current recombinant protein Transient Expression System
One of chief cell system.The scheme for turning technology in extensive wink, being presently mainly according to Florian M.Wurm et al. of Chinese hamster ovary celI,
The day before transfection carries out passage, and then fresh culture is changed in transfection same day centrifugation and to reach cell one higher close
Degree (2 × 106Cells/ml), then transfected with PEI (polyethyleneimine) as transfection reagent, after transfection cooling culture with
Suppress cell propagation, so as to maintain Cell viability.
Wherein, PEI is a kind of chemical transfection reagent, the cationic liposomal transfection reagent commonly used compared to scientific research institution,
Cost is relatively low, is more suitable for industrial production, and its cost performance can be more improved in larger recombinant protein transient expression production
High the characteristics of.The wherein PEI of 25kDa linear structures is because its applicability is wide, and transfection efficiency is generally good, so compared to other
The PEI of molecular weight (such as 40kDa) and other structures (such as dendroid), many documents all report more pro-gaze in linear using 25kDa
The PEI of structure is used as transfection reagent.
Epstein-Barr virus (Epstein-Barr virus) is also referred to as human herpesvirus 4, the epithelial cell of main infection people and immune
The B cell of system.There was only EBNA1 (Epstein-Barr nuclear in the whole latent infection phase of ebv infection
Antigen 1, eb nuclear antigen) there is expression all the time.There are DNA bonding pads, transcriptional activation domain, glycine-the third on EBNA1
Propylhomoserin duplicate block, nuclear localization signal sequence, DNA are combined and dimerization area.EBNA1 can (virus be multiple with the OriP on virus particle
Starting point processed) combine, in the case where nuclear localization signal guides the assistance with host cell cytosol transcription factor, carry virus particle and enter thin
Karyon, and combined by DNA bonding pads in chromosome corresponding site, virus particle is replicated with chromosome replication.
At the same time, the transcriptional activation domain on EBNA1 makes the effect that it plays trans-acting proteins, and its action site is also
OriP, plays the effect of its cis-acting elements, promotes the transcription of downstream gene.And the effect of Gly-Ala duplicate block
It is to stablize the conformation of EBNA1 mature proteins and avoid it from being easily degraded by proteases.
Therefore, the characteristics of could being expressed and its be combined with the OriP on virus particle using the EBNA1 of Epstein-Barr virus, builds one
Recombinant C HOK1 cell lines are planted, and design corresponding transient expression vector, cell transfecting and culture process, can be in restructuring
Remain to obtain higher expression quantity in the actual production of albumen transient expression, and technique simplicity is with low cost, it is adaptable to big rule
Mould is produced or small size high flux screening, is the problem of this area has to be solved.
The content of the invention
One of technical problems to be solved by the invention are there is provided a kind of construction method of recombinant C HOK1 cell lines, are somebody's turn to do
Recombinant C HOK1 cell lines can stablize expression truncated-type EBNA1 albumen (abbreviation EBNA1t), in the reality of recombinant protein transient expression
PEI can be used as transfection reagent in the production of border, expression quantity is high and technique simplicity is with low cost, it is adaptable to mass produce
Or small size high flux screening.
The two of the technical problems to be solved by the invention are that there is provided be suitable for recombinant C HOK1 cell lines in restructuring egg
Transient expression vector, cell transfecting and culture process in white transient expression, technique simplicity cost is low while expression quantity is high
It is honest and clean.
To solve one of above-mentioned technical problem, the present invention provides a kind of construction method of recombinant C HOK1 cell lines, Neng Gouwen
Surely expression truncated-type EBNA1 albumen (abbreviation EBNA1t), comprises the following steps：
(1) structure of EBNA1t expression vectors.
As shown in figure 1, EBNA1 full length proteins totally 641 amino acid, including following functions domain：(1) DNA bonding pads
1：33-89aa；(2) transcriptional activation domain：65-89aa；(3) Gly-Ala duplicate block：90-327aa；(4) DNA bonding pads
2,328-378aa；(5) nuclear localisation signal, 379-386aa；(6) DNA is combined and dimerization area, 459-604aa.
Behind the Gly-Ala duplicate block for removing EBNA1 albumen, the EBNA1 albumen (EBNA1t) of truncated-type is obtained.Through
Detect that EBNA1 albumen (EBNA1t) function of truncated-type is unaffected.
EBNA1t cDNA sequences such as SEQ ID NO:Shown in 1, it efficiently can express EBNA1t in CHOK1 cells
Albumen, and EBNA1t functions are unaffected.
EBNA1t cDNA sequences are cloned into pWX1.0 and pWX2.0 carriers, obtain pWX1.0-Z-EBNA1t and
PWX2.0-B-EBNA1t, as shown in Figure 2 and Figure 3.Wherein, pWX1.0 and pWX2.0 carriers respectively containing Zeocin and
Blasticidin resistant gene, the CHOK1 cell lines for screening stable expression EBNA1t.PWX1.0-Z-EBNA1t is expressed
Target gene EBNA1t and resistant gene Zeocin shares CMV promoter and TKpA tailing sequences, two expressing genes in carrier
Centre is separated with ECMV IRES sequences.Target gene EBNA1t uses CMV promoter in pWX2.0-B-EBNA1t expression vectors
With TKpA tailing sequences, resistant gene Blasticidin uses SV40 promoters and SVpA tailing sequences.
(2) transfection is screened with cell mass.
The CHOK1 host cells used in experiment are the cell of the culture that can be suspended in serum free medium through domestication
Strain, culture medium is CD CHO (Gibco 12490-003), and culture vessel is 125ml-5l shaking flasks, and condition is 36.5 DEG C, 85% wet
Degree, 6%CO2、150RPM。
Using cationic-liposome FreeStyle MAX Reagent (Invitrogen 16447100) by pWX1.0-
EBNA1t and pWX2.0-EBNA1t cotransfection CHOK1 host cells.
48 hours after transfection, seed cells into 96 orifice plates and cultivate, using containing antibiotic Blasticidin
(Invitrogen A11139-03) and Zeocin (Invitrogen R250-05) CD CHO Screening of Media.
After screening 2-3 weeks, the cell survived in 96 orifice plates is expanded respectively, if obtaining population of stem cells.
By the transient expression vector of desired monoclonal antibodies, transfected using PEI into each cell mass, harvesting after 10 days
Culture supernatant, assesses the expression quantity of desired monoclonal antibodies.Pick out desired monoclonal antibodies expression quantity highest cell mass use
In follow-up limiting dilution assay clone.
(3) limiting dilution assay is cloned.
Monoclonal is separated to from cell mass by limiting dilution assay, using containing antibiotic Blasticidin and
Zeocin CD CHO medium culture CHOK1-EBNA1t monoclonal cell strains.Similar, by the wink of desired monoclonal antibodies
When expression vector, transfected using PEI into each monoclonal cell strain, harvesting culture supernatant after 10 days, assess target Dan Ke
The expression quantity of grand antibody.Pick out desired monoclonal antibodies expression quantity highest CHOK1-EBNA1t monoclonal cell strains.
To solve the two of above-mentioned technical problem, the present invention, which provides a kind of recombinant C HOK1-EBNA1t cell lines, to be used to recombinate egg
The method of white transient expression, comprises the following steps：
(1) recombinant protein transient expression vector is built
Recombinant protein DNA sequence dna is cloned into the expression vector pWX4.0-OriP containing OriP cDNA sequences respectively,
For transiently transfecting.
The OriP cDNA sequences used in the present invention such as SEQ ID NO:Shown in 2, transient expression vector is as shown in Figure 4.
EBNA1t albumen is acted on the OriP elements on transient expression vector, so as to promote the expression of foreign gene.Under OriP sequences
Trip has CMV promoter and TKpA tailing sequences, there is some restriction enzyme positions between CMV promoter and TKpA tailing sequences
Point, in order to insert purpose recombinant protein gene.Recombinant protein refers mainly to the secreting types such as monoclonal antibody, Fc fusion proteins, enzyme
Recombinant protein.
(2) passage culture is carried out before transfecting
Before transfection, by CHOK1-EBNA1t cell lines diluted passage to fresh CD CHO culture mediums, Secondary Culture is carried out.
First 1 day of transfection, by 6-10 105Cell/ml connects fresh CD CHO culture mediums.Cultivation temperature is 36.5 DEG C.
(3) transfection and cell culture process
Recombinant protein transient expression vector is transfected into CHOK1- using the linear PEI of 25kDa (Polysciences 23966)
EBNA1t cell lines.Expression vector and PEI usage ratio are 1:2-1:7, per ml cell suspensions in the μ of 1-4 containing expression vector g.
Expression vector and PEI are added directly into cell.Cultivation temperature after transfection is reduced to 29-34 DEG C.
(4) cell conditioned medium is harvested
Harvesting culture supernatant after transfecting 10 days, assesses the expression quantity of recombinant protein.
In the method for the present invention, by building the Chinese hamster ovary celI strain of stable expression truncated-type EBNA1 albumen (EBNA1t), make
For the host cell of recombinant protein Transient Expression System, OriP elements are carried on expression vector, can be relatively low in use cost
When PEI is as transfection reagent, remains to keep compared with high transfection efficiency, cost can be saved, the recombinant protein wink of Chinese hamster ovary celI can be made again
When expression system can adapt to want scale and flux in the new medicament screen and preclinical study of industrial recombinant protein at present
Ask, but also expression quantity can be improved.
The beneficial effects of the present invention are：
1. the present invention carries out codon optimization to the cDNA sequence of the EBNA1 albumen (EBNA1t) of truncated-type, optimized
Such as SEQ ID NO of EBNA1t cDNA sequences afterwards:Shown in 1, it efficiently can express truncated-type in CHOK1 cells
EBNA1 albumen (EBNA1t), and EBNA1t functions are unaffected, the sequence can be used in building a kind of stable expression EBNA1 eggs
White Chinese hamster ovary celI strain, and for recombinant protein transient expression, more than 2.5 times expression quantity can be obtained.
2. the present invention constructs a kind of Chinese hamster ovary celI strain of stable expression EBNA1 albumen, the instantaneous table of recombinant protein can be used as
Up to the host cell of system.
3. recombinant protein sequence is cloned into the expression vector pWX4.0-OriP containing OriP by the present invention, then by carrier
In the Chinese hamster ovary celI strain of stable expression EBNA1t albumen for transiently transfecting the present invention, as recombinant protein transient expression, EBNA1t with
OriP combinations can promote the transient expression vector pWX4.0-OriP for carrying OriP elements to enter host cell nuclear and promote external source
The transcription of plasmid, so as to improve transfection efficiency.
4. the present invention is used for the method for recombinant protein transient expression, PEI can be used to transfect, technique simplicity is with low cost,
And expression quantity is high, is not only suitable for large-scale production and is also applied for small size high flux screening.
5. the recombinant protein instant expression method expression quantity of the present invention is high, generally 100-300mg/L is common
More than 2.5 times of CHOK1 cells.
Brief description of the drawings
Fig. 1 is the structure of EBNA1 albumen.
Fig. 2 is the expression vector pWX1.0-Z-EBNA1t of restructuring CHOK1 cells.
Fig. 3 is the expression vector pWX2.0-B-EBNA1t of restructuring CHOK1 cells.
Fig. 4 is recombinant protein transient expression vector pWX4.0-OriP collection of illustrative plates.
Embodiment
Clear, complete description is carried out to technical scheme below in conjunction with accompanying drawing, it is clear that described implementation
Example is a part of embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, the common skill in this area
The every other embodiment that art personnel are obtained on the premise of creative work is not made, belongs to the model that the present invention is protected
Enclose.
The recombinant C HOK1-EBNA1t cell lines that the present invention is built can be used for recombinant protein transient expression, and recombinant protein is main
Refer to the secreting type recombinant proteins such as monoclonal antibody, Fc fusion proteins, enzyme.It is specific real by taking the transient expression of monoclonal antibody as an example
Apply mode as follows：
(1) heavy chain and sequence of light chain of monoclonal antibody are cloned into the transient expression vector containing OriP elements respectively
In pWX4.0-OriP.Plasmid is extracted, concentration is determined, freezes standby in -80 DEG C.
(2) recovery CHOK1-EBNA1t cell lines (being built by the method for the present invention), uses CD CHO culture mediums
(Gibco 12490-003) cultivate, culture vessel be 125ml-5l shaking flasks, condition of culture be 36.5 DEG C, 85% humidity, 6%
CO2、150RPM.Passed on every 2-4 days.
(3) transfect first 1 day, by density 6-10 105Cells/ml is connected to fresh CD CHO culture mediums.
(4) recombinant protein transient expression vector is transfected using the linear PEI of 25kDa (Polysciences 23966)
CHOK1-EBNA1t cell lines.DNA and PEI usage ratio is 1:2-1:7, per ml cell suspensions in the μ g of 1-4 containing expression vector,
DNA and PEI are added directly into cell.Cultivation temperature after transfection is reduced to 29-34 DEG C.
(5) cell conditioned medium is harvested：Harvesting culture supernatant after transfecting 10 days, assesses the expression quantity of monoclonal.
The EBNA1 albumen (EBNA1t) of truncated-type is combined the transient expression vector that can promote to carry OriP elements with OriP
PWX4.0-OriP enters host cell nuclear and promotes the transcription of exogenous plasmid, so that improving transfection efficiency includes exogenous DNA plasmid
Enter efficiency and exogenous DNA plasmid transcription that the efficiency of endochylema, exogenous DNA plasmid enter nucleus through nuclear membrane through cell membrane
And translation efficiency, and improve exogenous gene expression amount.
Experiment tests the expression quantity of four kinds of monoclonal antibodies, such as table 1 in CHOK1 and CHOK1-EBNA1t cells respectively
It is shown.The expression quantity of CHOK1-EBNA1t cells is 2.5-3.5 times of CHOK1 cells.
Expression quantity (the unit of the monoclonal antibody of table 1:mg/mL)
| Monoclonal antibody | CHOK1 | CHOK1-EBNA1t | Multiple |
| Ab001 | 0.053 | 0.173 | 3.3 |
| Ab002 | 0.064 | 0.222 | 3.5 |
| Ab003 | 0.081 | 0.206 | 2.5 |
| Ab004 | 0.044 | 0.119 | 2.7 |
In summary, the various embodiments described above and accompanying drawing are only presently preferred embodiments of the present invention, not to limit this
The protection domain of invention, within the spirit and principles of the invention, any modification, equivalent substitution and improvements done etc. all should
Comprising within the scope of the present invention.
<110>Shanghai Yao Ming Bioisystech Co., Ltd, Wuxi Yao Ming Kants Biotechnology Ltd., the bright health of Suzhou medicine
Co., Ltd is examined in moral detection
<120>A kind of construction method of recombinant C HOK1 cell lines and its application
<130>CPC-NP-17-100423
<160> 2
<170> PatentIn version 3.4
<210> 1
<211> 1254
<212> DNA
<213>Artificial sequence
<400> 1
atgtccgatg aaggacccgg caccggaccc ggaaatggac tgggcgagaa gggcgataca 60
tccggccctg agggctccgg aggaagcgga cctcagagga ggggaggcga taatcacggc 120
cggggaaggg gcaggggccg gggccggggc ggcggccggc ccggcgctcc tggcggctcc 180
ggatccggcc ctaggcatag ggatggcgtg aggaggcctc agaagaggcc cagctgcatc 240
ggctgcaaag gcacccacgg cggcaccggc gctggcgctg gcgccggcgg cgccggcgct 300
ggcggcggcg gcaggggccg gggaggcagc ggcggcaggg gcaggggcgg cagcggcggc 360
aggggacggg gcggctccgg cggccggcgg ggccggggca gggagagggc caggggcgga 420
agcagggaaa gggctagggg caggggaagg ggaaggggag agaagaggcc ccggagccct 480
agctcccagt ccagcagcag cggcagccct cctaggaggc cccctcccgg caggaggccc 540
ttttttcacc ctgtgggcga ggctgactac ttcgagtacc accaggaggg cggacctgac 600
ggagagcctg atgtgcctcc tggcgccatc gaacagggcc ctgctgacga tcctggcgag 660
ggaccttcca ccggacctag gggccaggga gacggaggaa ggaggaagaa aggcggctgg 720
ttcggaaaac ataggggcca aggcggcagc aaccccaagt ttgagaacat cgccgaaggc 780
ctgagggctc tgctggctcg gagccatgtg gagcggacca ccgatgaggg cacctgggtg 840
gctggcgtgt tcgtgtacgg cggctccaag accagcctgt acaacctcag gaggggaacc 900
gccctggcta tccctcagtg taggctgaca cccctgagcc ggctgccctt cggaatggcc 960
cctggacctg gacctcagcc tggccctctg agggagagca tcgtgtgcta cttcatggtg 1020
ttcctgcaga cccacatttt cgccgaggtg ctgaaggacg ccatcaagga cctggtgatg 1080
accaagcctg cccctacatg caacatccgg gtgaccgtgt gcagcttcga tgacggcgtg 1140
gatctgcccc cttggttccc tcccatggtg gagggagccg ctgctgaggg agacgatggc 1200
gacgacggcg atgaaggcgg agatggcgat gagggagagg agggccagga atga 1254
<210> 2
<211> 913
<212> DNA
<213> OriP
<400> 2
ccgcggactt aagcccgctc gaggacacgc gtctgatatc aaccctaaac gggtagcata 60
tgcttcccgg gtagtagtat atactatcca gactaaccct aattcaatag catatgttac 120
ccaacgggaa gcatatgcta tcgaattagg gttagtaaaa gggtcctaag gaacagcgat 180
gtaggtgggc gggccaagat aggggcgcga ttgctgcgat ctggaggaca aattacacac 240
acttgcgcct gagcgccaag cacagggttg ttggtcctca tattcacgag gtcgctgaga 300
gcacggtggg ctaatgttgc catgggtagc atatactacc caaatatctg gatagcatat 360
gctatcctaa tctatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat 420
gctatcctaa tctatatctg ggtagtatat gctatcctaa tttatatctg ggtagcatag 480
gctatcctaa tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat 540
gctatcctaa tctgtatccg ggtagcatat gctatcctaa tagagattag ggtagtatat 600
gctatcctaa tttatatctg ggtagcatat actacccaaa tatctggata gcatatgcta 660
tcctaatcta tatctgggta gcatatgcta tcctaatcta tatctgggta gcataggcta 720
tcctaatcta tatctgggta gcatatgcta tcctaatcta tatctgggta gtatatgcta 780
tcctaattta tatctgggta gcataggcta tcctaatcta tatctgggta gcatatgcta 840
tcctaatcta tatctgggta gtatatgcta tcctaatctg tatccgggta gcatatgcta 900
tcctcatgga tcc 913
Claims (11)
1. a kind of construction method for the recombinant C HOK1 cell lines that can stablize expression truncated-type EBNA1 albumen, including following step
Suddenly：
(1) structure of EBNA1t expression vectors
It is EBNA1t to define truncated-type EBNA1 albumen；Will be such as SEQ ID NO:Sequence shown in 1 is cloned into pWX1.0 and pWX2.0 and carried
In body, pWX1.0-Z-EBNA1t and pWX2.0-B-EBNA1t are obtained；
(2) transfection is screened with cell mass
Using cationic-liposome by pWX1.0-Z-EBNA1t and pWX2.0-B-EBNA1t cotransfection CHOK1 host cells；
48 hours after transfection, seed cells into 96 orifice plates and cultivate, using containing antibiotic Blasticidin and Zeocin
CD CHO Screening of Media；
After screening 2-3 weeks, the cell survived in 96 orifice plates is expanded respectively, if obtaining population of stem cells；
By the transient expression vector of desired monoclonal antibodies, transfected using PEI into each cell mass, harvesting culture after 10 days
Supernatant, assesses the expression quantity of desired monoclonal antibodies, picks out after desired monoclonal antibodies expression quantity highest cell mass is used for
Continuous limiting dilution assay clone；
(3) limiting dilution assay is cloned
Monoclonal is separated to from cell mass by limiting dilution assay；Pick out desired monoclonal antibodies expression quantity highest
CHOK1-EBNA1t monoclonal cell strains.
2. the method as shown in claim 1, it is characterised in that in the step (1), SEQ ID NO:Sequence shown in 1 is excellent
EBNA1t cDNA sequences after change, are the Gly-Ala duplicate blocks by removing EBNA1 albumen：After 90-327aa, obtain
To the EBNA1 albumen of truncated-type.
3. the method as shown in claim 1, it is characterised in that in the step (1), pWX1.0 and pWX2.0 carriers contain respectively
There are Zeocin and Blasticidin resistant gene.
4. the method as shown in claim 1, it is characterised in that in the step (2), culture vessel is 125ml-5l shaking flasks,
Condition is 36.5 DEG C, 85% humidity, 6%CO2、150RPM。
5. the method as described in claim 1, it is characterised in that in the step (3), using containing antibiotic
Blasticidin and Zeocin CD CHO medium culture CHOK1-EBNA1t monoclonal cell strains；Desired monoclonal is resisted
The transient expression vector of body, is transfected into each monoclonal cell strain using PEI, harvesting culture supernatant after 10 days, assesses mesh
Mark the expression quantity of monoclonal antibody.
6. a kind of truncated-type EBNA1 Protein cDNA Sequences, it is characterised in that such as SEQ ID NO:Shown in 1.
7. a kind of expression vector pWX1.0-Z-EBNA1t of truncated-type EBNA1 albumen, it is characterised in that be by will be such as SEQ
ID NO:Sequence shown in 1 is cloned into pWX1.0 carriers and obtained, and the pWX1.0 contains Zeocin resistant gene.
8. a kind of expression vector pWX2.0-B-EBNA1t of truncated-type EBNA1 albumen, it is characterised in that be by will be such as SEQ
ID NO:Sequence shown in 1 is cloned into pWX2.0 carriers and obtained, and the pWX2.0 contains Blasticidin resistant gene.
9. a kind of recombinant C HOK1-EBNA1t cell lines are used for the application of recombinant protein transient expression, it is characterised in that described
CHOK1-EBNA1t cell lines are obtained by such as any one of claim 1-5 methods describeds, and the application comprises the following steps：
(1) recombinant protein transient expression vector is built
Recombinant protein sequence is cloned into the expression vector pWX4.0-OriP containing OriP sequences respectively, obtains being used for instantaneously
The recombinant protein transient expression vector of transfection；The OriP sequences such as SEQ ID NO:Shown in 2；
(2) passage culture is carried out before transfecting
Before transfection, by CHOK1-EBNA1t cell lines diluted passage to fresh CD CHO culture mediums, Secondary Culture is carried out；
(3) transfection and cell culture process
Recombinant protein transient expression vector transfection procedure (2) Secondary Culture that step (1) is obtained is obtained using 25kDa linear PEI
The CHOK1-EBNA1t cell lines obtained；Expression vector and PEI usage ratio are 1:2-1:7, table is contained in every milliliter of cell suspension
Up to carrier 1-4 μ g；
(4) harvesting supernatant, assesses the expression quantity of recombinant protein.
10. application as claimed in claim 9, it is characterised in that in step (2), is transfected first 1 day, by cell according to 6-
10105Cells/ml passes on fresh CD CHO culture mediums；Cultivation temperature is 36.5 DEG C.
11. application as claimed in claim 9, it is characterised in that in step (3), the cultivation temperature after transfection is 29-34 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710172903.1A CN106987559A (en) | 2017-03-22 | 2017-03-22 | A kind of construction method of recombinant C HOK1 cell lines and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710172903.1A CN106987559A (en) | 2017-03-22 | 2017-03-22 | A kind of construction method of recombinant C HOK1 cell lines and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106987559A true CN106987559A (en) | 2017-07-28 |
Family
ID=59411711
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710172903.1A Pending CN106987559A (en) | 2017-03-22 | 2017-03-22 | A kind of construction method of recombinant C HOK1 cell lines and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106987559A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129365A (en) * | 2019-05-22 | 2019-08-16 | 北京景达生物科技有限公司 | A kind of method and its application of efficient stable transient expression recombinant protein |
| CN110804625A (en) * | 2019-11-21 | 2020-02-18 | 安徽大学 | Method for increasing production of therapeutic antibody by over-expressing PTEN C124S in CHO (Chinese hamster ovary) cells |
| WO2020034986A1 (en) * | 2018-08-14 | 2020-02-20 | Wuxi Biologics (Shanghai) Co., Ltd. | Transcriptional regulatory element and its use in enhancing the expression of heterologous protein |
| CN111406105A (en) * | 2018-11-02 | 2020-07-10 | 上海药明生物技术有限公司 | Enhanced perfusion cell culture method with continuous harvest and no cell discharge |
| CN114107380A (en) * | 2021-11-05 | 2022-03-01 | 上海药明生物技术有限公司 | CHO-S.attp recombinant cell strain and construction method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006096989A2 (en) * | 2005-03-17 | 2006-09-21 | National Research Council Of Canada | Expression vectors containing a truncated epstein barr nuclear antigen 1 lacking the gly-gly-ala domain for enhanced transient gene expression |
| CN1910287A (en) * | 2003-09-09 | 2007-02-07 | 艾塞特生物技术有限公司 | Rodent expression systems utilising polyoma virus and epstein barr virus sequences |
| WO2009137911A1 (en) * | 2008-05-15 | 2009-11-19 | National Research Counsil Of Canada | Process, vectors and engineered cell lines for enhanced large-scale transfection |
| CN103205407A (en) * | 2008-12-09 | 2013-07-17 | 哈洛齐梅公司 | Extended soluble ph20 polypeptides and uses thereof |
| CN105779501A (en) * | 2014-12-26 | 2016-07-20 | 上海药明康德新药开发有限公司 | Method for carrying out transient expression production on recombinant protein by adopting CHO (Chinese Hamster Ovary) host cells |
-
2017
- 2017-03-22 CN CN201710172903.1A patent/CN106987559A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1910287A (en) * | 2003-09-09 | 2007-02-07 | 艾塞特生物技术有限公司 | Rodent expression systems utilising polyoma virus and epstein barr virus sequences |
| WO2006096989A2 (en) * | 2005-03-17 | 2006-09-21 | National Research Council Of Canada | Expression vectors containing a truncated epstein barr nuclear antigen 1 lacking the gly-gly-ala domain for enhanced transient gene expression |
| WO2009137911A1 (en) * | 2008-05-15 | 2009-11-19 | National Research Counsil Of Canada | Process, vectors and engineered cell lines for enhanced large-scale transfection |
| CN103205407A (en) * | 2008-12-09 | 2013-07-17 | 哈洛齐梅公司 | Extended soluble ph20 polypeptides and uses thereof |
| CN105779501A (en) * | 2014-12-26 | 2016-07-20 | 上海药明康德新药开发有限公司 | Method for carrying out transient expression production on recombinant protein by adopting CHO (Chinese Hamster Ovary) host cells |
Non-Patent Citations (3)
| Title |
|---|
| 张钧田 等: "《现代药理试验方法 上》", 31 July 2012, 中国协和医科大学出版社 * |
| 钱凯先编著: "《克隆风云 现代生命科学论著》", 31 March 1999, 浙江大学出版社 * |
| 陶维红 等: "CHO细胞株开发技术策略探讨", 《生物技术进展》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020034986A1 (en) * | 2018-08-14 | 2020-02-20 | Wuxi Biologics (Shanghai) Co., Ltd. | Transcriptional regulatory element and its use in enhancing the expression of heterologous protein |
| US11254952B2 (en) | 2018-08-14 | 2022-02-22 | Wuxi Biologies Ireland Limited | Transcriptional regulatory element and its use in enhancing the expression of heterologous protein |
| CN111406105A (en) * | 2018-11-02 | 2020-07-10 | 上海药明生物技术有限公司 | Enhanced perfusion cell culture method with continuous harvest and no cell discharge |
| CN110129365A (en) * | 2019-05-22 | 2019-08-16 | 北京景达生物科技有限公司 | A kind of method and its application of efficient stable transient expression recombinant protein |
| CN110129365B (en) * | 2019-05-22 | 2023-05-05 | 北京景达生物科技有限公司 | Method for high-efficiency stable instantaneous expression of recombinant protein and application thereof |
| CN110804625A (en) * | 2019-11-21 | 2020-02-18 | 安徽大学 | Method for increasing production of therapeutic antibody by over-expressing PTEN C124S in CHO (Chinese hamster ovary) cells |
| CN114107380A (en) * | 2021-11-05 | 2022-03-01 | 上海药明生物技术有限公司 | CHO-S.attp recombinant cell strain and construction method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106987559A (en) | A kind of construction method of recombinant C HOK1 cell lines and its application | |
| CN108504657B (en) | The method for knocking out HEK293T cell KDM2A gene using CRISPR-CAS9 technology | |
| CN107828738A (en) | A kind of dnmt rna deficiency Chinese hamster ovary celI system and preparation method and application | |
| CN109136193A (en) | NW_006884764-1 stablizes the application of expression protein in a kind of CHO cell genome | |
| CN114058625B (en) | Site for stably expressing protein in CHO cell gene NW _003613781.1 and application thereof | |
| CN101215577A (en) | General purpose method for preparing integration type cell immortalization vector | |
| CN114085841A (en) | Site for stably expressing protein in CHO cell gene NW _003614092.1 and application thereof | |
| CN110981968B (en) | Fusion protein containing rabies virus G protein, preparation method, application and vaccine thereof | |
| CN109321604A (en) | NW_006882077-1 stablizes the application of expression protein in a kind of CHO cell genome | |
| CN106497952A (en) | A kind of based on the film ankyrin of CD86, its preparation method and application | |
| Gu et al. | Establishment and characterization of an immortalized renal cell line of the Chinese tree shrew (Tupaia belangeri chinesis) | |
| CN102321655A (en) | Pseudo-attP site based integrated general-purpose expression vector and construction method and application thereof | |
| CN113969283B (en) | Site for stably expressing protein in CHO cell gene NW _003613756.1 and application thereof | |
| US4687737A (en) | Mammalian suppressor genes | |
| CN109207432A (en) | NW_006883358-1 stablizes the application of expression protein in a kind of CHO cell genome | |
| CN109295093A (en) | NW_006882456-1 stablizes the application of expression protein in a kind of CHO cell genome | |
| CN105481984A (en) | Transposase for efficiently mediating exogenous gene integration, and use thereof | |
| CN108070609A (en) | By the use of trichoderma reesei as the method for host expresses recombinant protein | |
| CN101693902B (en) | Construction method of conditional gene knockout carrier | |
| Suarez-Patino et al. | Transient expression of rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells | |
| CN103352050B (en) | Method for improving cell transfection efficiency through utilization of bacterial artificial chromosome homologous recombination | |
| CN109402096B (en) | AID enzyme mutant and application thereof | |
| CN104946685A (en) | Method for in vitro preparation of pig dendritic cells by using recombinant protein | |
| CN101397573A (en) | Retroviral vector for expression of glial cell line-derived neurotrophic factor and use thereof | |
| CN106867966B (en) | Vertebrate cell line stably expressing core alpha (1,3) fucosyltransferase and preparation method 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 | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 200131 701, 7 floor, 2 Hua Jing Road, Pudong New Area, Shanghai. Applicant after: SHANGHAI YAOMING BIOTECHNOLOGY CO., LTD. Applicant after: Wuxi Yaoming Biotechnology Co., Ltd. Applicant after: SUZHOU YAOMING KANGDE INSPECTION TESTING CO., LTD. Address before: 200131 701, 7 floor, 2 Hua Jing Road, Pudong New Area, Shanghai. Applicant before: SHANGHAI YAOMING BIOTECHNOLOGY CO., LTD. Applicant before: WUXI APPTEC BIOPHARMACEUTICALS CO., LTD. Applicant before: SUZHOU YAOMING KANGDE INSPECTION TESTING CO., LTD. |
|
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 200131 701, 7 floor, 2 Hua Jing Road, Pudong New Area, Shanghai. Applicant after: SHANGHAI YAOMING BIOTECHNOLOGY CO., LTD. Applicant after: Wuxi Yaoming Biotechnology Co., Ltd. Applicant after: Suzhou Pharmacopoeia Testing and Inspection Co., Ltd. Address before: 200131 701, 7 floor, 2 Hua Jing Road, Pudong New Area, Shanghai. Applicant before: SHANGHAI YAOMING BIOTECHNOLOGY CO., LTD. Applicant before: Wuxi Yaoming Biotechnology Co., Ltd. Applicant before: SUZHOU YAOMING KANGDE INSPECTION TESTING CO., LTD. |
|
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170728 |