CN104087612A - Method for fast screening mammal cell strain having high exogenous protein expression level - Google Patents
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Abstract
The invention relates to a method for fast screening a mammal cell strain having a high exogenous protein expression level. The method utilizes a novel high-expression carrier, realizes efficient expression of a desired protein in an appropriate mammal host cell, and realizes fast screening of a cell strain having a high desired protein expression level by a unique screening method. The method greatly reduces time and a cost of screening of the cell strain having a high desired protein expression level and substantially improves an expression level of the exogenous protein in the mammal host cell.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method that rapid screening is stablized high yield mammalian cell strain.Utilize screening method of the present invention can obtain fast the stable mammalian cell strain of high expression level foreign protein.
Background technology
Along with the development of biological medicine technology, treatment is widely used in clinical practice with recombinant protein (comprising antibody).Many curative recombinant proteins will keep its biological activity often to need specific posttranslational modification, and as glycosylation etc., and prokaryotic organism or rudimentary eukaryotic cells expression system lack complicated posttranslational modification function.Along with popularizing gradually of glycoprotein analog medicine, mammalian expression system especially Chinese hamster ovary celI (Chinese hamster ovary, Chinese hamster ovary cell) expression system is just becoming the main host cell that recombinant protein medicine is produced.At present existing various exogenous genes is as human histiotype plasminogen activator, erythropoietin (Erythropoietin, abbreviation EPO), blood coagulation factor VIII and monoclonal antibody CD20 etc. are expressed and are produced in mammalian cell, said medicine is all gone on the market, and each medicine annual sales amount is over 1,000,000,000 dollars.
Yet, because mammalian cell growth is slow, medium component is complicated, growth conditions is harsh, and the factor such as the stable high yielding cell sarain that exogenous protein expression level is low, target protein is expressed in screening takes time and effort, cell cultures cost is high, technical sophistication, cause that the cost that utilizes mammalian cell to produce protein drug is higher, the cycle is long.Rapid screening is stablized the Cell engineering strain of high yield, is one of Main Bottleneck of current recombinant protein medicine research and production.
In order to obtain the high expression level stable cell line of expressing target protein, need to apply expression vector and target gene is transfected into mammalian host cell and is incorporated on host cell chromosome, along with the transcription and translation of host cell, target gene is able to effective expression.Therefore, the key element that obtains high expression level mammalian cell strain comprises expression vector, host cell, transfection and screening method, wherein expression vector and cell line screening method be target protein be able to that stability and high efficiency expresses greatest factor.
Traditional mammalian cell expression vector comprises four expression cassettes conventionally, be respectively used to express target gene, mammalian cell resistance screening marker gene (as bleomycin, Zeocin, abbreviation ZEO), amplification selected marker is (as Tetrahydrofolate dehydrogenase, dyhidrofolate reductase, be called for short DHFR) and in intestinal bacteria, copy amplified gene, each expression cassette contains separately independently promotor and terminator, is positioned at the upstream and downstream of above-mentioned expressing gene.Expression vector enters after cell, and proteins encoded produces by transcribing with translating mechanism and necessary posttranslational modification of cell.For mammalian host cell, the integration of exogenous array will inevitably have influence on the normal growth of host cell, and host cell can be resisted by a series of mechanism the integration of foreign gene.Because traditional mammalian cell expression vector contains 4 expression cassettes, carrier is generally greater than 8kb, is unfavorable for that exogenous origin gene integrator is to host cell chromosome, increases the burden of host cell and causes the injury of foreign gene to host cell.The size of therefore reducing carrier, significant for the high efficiency stable expression of target gene.In addition, due to the nonspecific integration of foreign gene, carrier is crossed conference and is caused its each element to be incorporated into the different positions of host cell gene group, brings very large inconvenience to the screening operation in later stage.For example, the cell obtaining with resistance screening is likely the cell of only having integrated and having expressed resistance screening gene, and does not express the cell of target protein.Therefore reducing carrier size also helps to reduce and screens false-positive probability.
Rapid screening method is the key factor that obtains high yield stable expression cell strain.Conventional screening method is to utilize single selection markers to screen the stable cell line that contains target gene, single selection markers is generally resistance marker, as ZEO mark, specifically, selection markers gene and goal gene are connected in identical carrier, then be transfected in host cell, realize the co expression of goal gene and marker gene, in conventional screening method, marker gene is close with target gene expression level, what likely screen is the cell of presentation markup gene, rather than expresses the cell of target gene.Thereby conventional screening method need to be stablized monoclonal cell to high expression level at thousands of cell screenings, obtain the time of the process stablize overexpression cell line more than at least needing half a year, expend a large amount of time and cost, restricted the scale operation of recombinant protein medicine.
Summary of the invention
The present invention builds by Protocols in Molecular Biology and optimizes mammalian cell new expression vector, utilize expression vector of the present invention that target gene is transfected into suitable host cell, and by unique screening method, obtain the engineering cell strain of high expression level target gene.The present invention can screen the engineering cell strain of high expression level foreign protein quickly and efficiently, and the high efficiency stable expression of realize target albumen in mammalian cell saved protein drug cost of development and time.
An object of the present invention is to provide a kind of efficient novel mammalian cell expression vector.Specifically, expression vector of the present invention is a kind of novel mammalian expression vector that utilizes Protocols in Molecular Biology to build and optimize.This expression vector is only containing single expression cassette, do not contain any target protein unnecessary redundant sequence in mammalian cell expression system, utilize the high efficient expression of a promotor realize target gene and the reduction of marker gene to express, mammalian cell expression vector of the present invention than traditional expression vector to 3kb when young.Simultaneously, in expression vector of the present invention, in the middle of marker gene and target gene, introduced IRES (Internal ribosome entry site, internal ribosome entry site) sequence, this sequence can weaken the expression of downstream marker gene, thereby when marker gene is expressed, guarantee that target gene is able to the expression of efficient stable.
Specifically, expression vector of the present invention is the optimum combination of a plurality of genes or gene fragment critical elements, and described expression vector is comprised of following element or sequence, and preferably with following order, arranges:
A) strong promoter
B) target gene cloning site
C) synthetic IRES sequence
D) amplification label gene
E) FMDV IRES sequence
F) synthetic intestinal bacteria EM7 element
G) chimeric selection markers gene
H) transcription terminator
I) one is enabled in the initial PUC sequence copying in intestinal bacteria.
A described strong promoter is viral promotors, as CMV (cytomegalovirus, cytomegalovirus), SV40 (Simian virus40, vacuolating virus of monkey 40), RSV (Rous sarcoma virus, sarcoma virus), MLV (Murine leukovirus, mouse leukaemia virus), MPSV (myeloproliferative sarcoma virus, and/or the combination of non-viral promotor (as people's ferritin heavy chain core promoter) and other regulating and controlling sequences bone marrow proliferation sarcoma virus).Preferably strong promoter is, CMV promotor, and its nucleotide sequence is as shown in SEQ ID NO:1.
Described target gene cloning site is, for a plurality of restriction endonuclease sites of cloned target gene.
Described synthetic IRES sequence is the part in 5 ' untranslated region of a plurality of repetitions of GTX homeodomain protein known in the art, has the function of IRES, and its nucleotide sequence is as shown in SEQ ID NO:3.
Described amplification label gene is DHFR sequence or GS sequence (Glutaminesynthetase, glutamine synthetase) known in the art.DHFR catalysis dihydrofolic acid be converted into tetrahydrofolic acid (THFA).Methotrexate (MTX) suppresses Tetrahydrofolate dehydrogenase, so transfection DHFR defective type host cell, can form the resistance to MTX by amplification DHFR gene.
Described FMDV IRES sequence is known in the art from the foot and mouth disease virus IRES sequence of (Foot and mouth disease virus is called for short FMDV), and its nucleotide sequence is as shown in SEQ ID NO:2.
The EM7 element that described synthetic intestinal bacteria EM7 element is synthetic, this element only contains 66 base sequences, and its nucleotide sequence is as shown in SEQ ID NO:4.
The combination that described chimeric selection markers gene is a plurality of selectivity resistant maker genes, resistant gene comprises anti-ZEO, PUR (puromycin, tetracycline), HYP (Hygromycin, Totomycin) or any one fluorescent mark gene, preferably comprise GFP (Green Fluorescent Protein, green fluorescent protein), RFP (Red Fluorescent Protein, red fluorescent protein).
Described transcription terminator is terminator sequence known in the art, as BGH (bovine growth hormone, Trobest) terminator, SV40 terminator, HSV TK (Herpes simplex virus-thymidine kinase, herpes simplex virus thymidine kinase gene) terminator.
Described one to be enabled in the initial PUC sequence copying in intestinal bacteria be the known in the art initial PUC homing sequence copying in intestinal bacteria that is enabled in.
Another object of the present invention is to provide a kind of method of mammalian cell strain of rapid screening high expression level foreign protein, and the method comprises the following steps:
A) utilize the novel efficient expression vector of the invention described above that target gene is transfected into host cell;
B) utilize the screening method rapid screening of uniqueness of the present invention to obtain the stable cell line of high expression level target protein.
Described expression vector, containing single expression cassette, is only the optimum combination of a plurality of genes or gene fragment critical elements, and its structure is identical with the expression vector structure of the invention described above.
Described host cell is selected from CHO, NS0, HEK293, Sp2/0, preferably Chinese hamster ovary celI.
Described transfection method is that described transfection method comprises calcium phosphate method, and electricity turns method (electroporation), liposome transfection (phospholipids), and preferred transfection method is that electricity turns method transfection.
Described rapid screening method is to utilize chimeric selection markers to carry out the screening of high expression level stable cell line.Chimeric selection markers can be the arbitrary combination of the resistant genes such as HYP, ZEO and PUR and DHFR, GS or GFP.The combination (ZEO-GFP) that preferred chimeric selection markers is ZEO and GFP, its nucleotide sequence is as shown in SEQ ID NO:5.
Screening method of the present invention has adopted chimeric selection markers simultaneously, can greatly improve screening efficiency, specifically, the cell strain that utilizes conventional screening methods to obtain high and stable yields expression target protein needs 6-9 month, and the screening method of employing uniqueness of the present invention only needs 8-9 week just can obtain the cell strain of high expression level target protein.
The method of rapid screening high and stable yields cell strain of the present invention is compared with conventional screening method, has the following advantages and effect:
Expression vector of the present invention only contains single mammalian cell expression frame, and between target gene and marker gene, introduced synthetic optimization IRES sequence, greatly reduce false positive clone's probability, and significantly improve the expression level of target gene in host cell.
Based on above-mentioned expression vector, the present invention has adopted unique screening method, and ordinary method need to be 10
3in the cell of geometric multiplicity, screen the cell clone of expressing target gene, and the present invention only need be from 10
2in the cell of geometric multiplicity, just can screen the cell clone of high expression level target gene.Particularly chimeric GFP fluorescent mark, can use flow cell sorter to carry out high-throughput sorting according to fluorescence intensity, use fluorescent microscope further to confirm positive expression clone, simplify screening step, shorten screening time, thereby greatly improved screening efficiency.In other words, the optimal expression level of utilizing conventional expression system to obtain recombinant protein in mammalian cell needs 6-9 month, and utilizes expression system of the present invention only to need 8-9 week just can obtain the cell strain of high expression level target protein.
Accompanying drawing explanation
Accompanying drawing has been emphasized principle of the present invention, not necessarily proportional.
The mammalian cell expression vector schematic diagram that Fig. 1: embodiment 1 builds.
A) traditional mammalian cell expression vector pA.Tradition expression vector contains four independently expression cassettes, and putting in order of each element is as follows: (1) CMV promotor; (2) target gene cloning site; (3) BGH terminator; (4) EF1-α promotor; (5) DHFR amplification gene sequence; (6) HSV TK terminator; (7) SV40 promotor; (8) ZEO resistant gene; (9) SV40 terminator; (10) PUC replication orgin; (11) intestinal bacteria skeleton and ammonia benzyl resistant gene (Amp
r).
B) expression vector pB of the present invention.Expression vector of the present invention only comprises single expression cassette, adopts different IRES sequences to connect between different expressing genes.Putting in order of each element of expression vector pB is as follows: (1) CMV promotor; (2) target gene cloning site; (3) synthetic GTX IRES sequence; (4) DHFR amplification gene; (5) FDMV IRES sequence; (6) EM7 element; (7) ZEO
rthe chimeric selection markers gene of-GFP; (8) SV40 terminator; (9) PUC replication orgin.
Western Bloting result figure in Fig. 2: embodiment 2.Compared containing the expression system A (pA-EPO) of EPO gene and the level of expression system B (pB-EPO) stably express target gene.
In Fig. 3: embodiment 2, obtain stable transfection containing the pA (pA-EPO) of EPO gene and the expression amount comparison diagram of pB (pB-EPO) cell strain.
The result figure of Western Bloting in Fig. 4: embodiment 5.Compared containing the expression system A (pA-tPA) of tPA gene and the level of expression system B (pB-tPA) stably express target gene.
In Fig. 5: embodiment 5, obtain stable transfection containing the pA (pA-tPA) of tPA gene and the expression amount comparison diagram of pB (pB-tPA) cell strain.
Embodiment
Embodiment 1: the structure of mammalian cell expression vector
Method: pass through Protocols in Molecular Biology, if restriction enzyme digestion, DNA connection, intestinal bacteria conversion and colony screening are according to the < < molecular cloning experiment guide > > (third edition, Huang Peitang translates, Science Press) etc. standard method build traditional expression vector pA and expression vector pB of the present invention.Concrete operation method is as follows:
The structure of 1.1 traditional expression vector pA: the pcDNA3.1 of the Invitrogen company carrier of take is template.
The fusion sequence (1.8kb) of synthetic promotor SV40, DHFR, terminator sv40 and other regulating and controlling sequences also adds respectively the restriction enzyme site of speI and NaeI at its end.Adopt T4DNA ligase enzyme to be connected to the pcDNA3.1 through speI and NaeI double digestion this synthetic fusion sequence, by intestinal bacteria, transform, and carry out mono-clonal screening and obtain traditional expression vector pA.
The structure of 1.2 expression vector pB of the present invention: utilize restriction enzyme BgIII and ScaI double digestion to remove
Frame sequence in intestinal bacteria on pcDNA3.1 carrier (1.4kb), will connect with T4DNA ligase enzyme after sticky end polishing with Klenow enzyme again.Use restriction enzyme to remove BGH terminator, SV40 promoter sequence (1.5kb), the fusion dna sequence that synthetic contains GTX IRES, DHFR, FWDV IRES E7 fusion sequence and chimeric selectable marker gene ZEO-GFP simultaneously, the carrier of enzyme being cut with T4DNA ligase enzyme is connected with the fusion sequence of synthetic.By intestinal bacteria, transform with colony screening and obtain expression vector pB of the present invention.
Result: Fig. 1 has shown the structure iron (size: 7.4kb) and the structure iron (size: 4.5kb) of expression vector pB of the present invention of traditional mammalian cell expression vector pA.
Tradition expression vector contains four independently expression cassettes.Putting in order of each element of expression vector pA is as follows:
A) CMV promotor;
B) target gene cloning site;
C) BGH terminator;
D) EF1-α promotor;
E) DHFR amplification gene sequence;
F) HSV TK terminator;
G) SV40 promotor;
H) ZEO resistant gene;
I) SV40 terminator;
J) PUC replication orgin;
K) intestinal bacteria frame sequence and ammonia benzyl resistant gene (Amp
r) sequence
Expression vector pB of the present invention only, containing single expression cassette, is comprised of following element or sequence, and arranges in the following order:
A) CMV promotor;
B) target gene cloning site;
C) synthetic GTX IRES sequence
D) DHFR amplification label gene;
E) FMDV IRES sequence;
F) EM7 element
G) ZEO
rthe chimeric selection markers gene of-GFP;
H) SV40 terminator;
I) PUC replication orgin.
Result: with the traditional mammalian cell expression vector comparison that contains a plurality of expression cassettes, expression vector pB of the present invention is only containing an expression cassette, only use a CMV promotor, deleted frame sequence and other unnecessary promotors and the terminator sequence in intestinal bacteria simultaneously, than conventional carriers to 3kb when young.
Embodiment 2: the stable cell line of rapid screening high expression level epo protein
2.1. build the expression vector that contains target gene: according to the encoding sequence of the existing sequence information synthetic of NCBI EPO, and by introducing corresponding restriction enzyme site (NheI and HindIII), be cloned in the expression vector pA and pB of embodiment 1 structure, obtain the expression plasmid pA-EPO and the pB-EPO that contain target gene.
2.2.DNA transfection is to CHO-dhfr
-suspension cell strain: by above-mentioned expression plasmid pA-EPO and the pB-EPO that contains target gene, and expression vector contrast pA and pB are transfected into respectively the Chinese hamster ovary celI (CHO-dhfr of DHFR enzyme defect
-) in.CHO-dhfr
-cell is suspension culture, and during transfection, cell density is 1.5 * 10
6/ ml, transfection volume is 30ml.Transfection method operates according to Lipofectamine2000 (purchased from Invitrogen) operation instruction.
2.3. the screening of stably express EPO cell strain:
Concrete grammar is as follows:
(1) by above-mentioned DNA transfection to CHO-dhfr
-after cell, shake-flask culture 48 hours, samples respectively observation of cell vigor, and vigor is greater than 90% can carry out kind of a plate.With the resuspended cell after centrifugal of fresh selection substratum (containing 100 μ g/ml Zeocin), with 1000-5000/hole kind in 96 orifice plates.The growth of 20 days visible significantly cell clones of transfection.
(2) select transfection respectively whole cell clones of 4 kinds of DNA in 96 other orifice plates, with the suspension medium containing resistance, cultivate 3 days, get supernatant and with ELISA, measure the expression of EPO in 96 orifice plates, with this, calculate positive colony rate.
(3) supernatant of having cultivated cell clone 96 orifice plates of 4 kinds of DNA is collected respectively and mix after be Western Blotting, for detection of 4 kinds of transfections the cell expressing of different DNA.
(4) according to ELISA measurement result, the cell clone of the highest OD reading of selected respectively 5-10 transfection pA-EPO and pB-EPO expression plasmid increases, and during amplification, with the suspension medium containing resistance, cultivates.When each clonal expansion to cell count is greater than 1 * 10
7when individual, in order to improve the copy number of goal gene and the stably express of target gene, each clone adopts MTX pressurization screening method.
(5) transfection the gradient MTX for cell clone of pA-EPO (50nM, 500nM, 1 μ M, 2 μ M, 3 μ M, 4 μ M, the 5 μ M) screening of progressively pressurizeing, the cell density that is at every turn inoculated in 96 orifice plates is every hole 100-500, the screening time of each gradient is 10-15 days, and detect compared with the 1-2 of high expression level clone and pressurize for next gradient MTX with ELISA, finally obtain the cell strain of stably express.
(6) transfection the cell clone of pB-EPO first with flow cytometry, filter out 5% the brightest cell of fluorescence, with the 5 μ M MTX screening of pressurizeing, the cell density that is inoculated in 96 orifice plates is every hole 100-500, screening time is 10-15 days, and detect the cell strain candidate as stably express compared with the 1-2 of high expression level clone with ELISA, final by relatively obtaining final engineering cell strain.
(7) two kinds of transfections containing the cell clone of EPO expression plasmid conservation 5 * 10 respectively
7after total cellular score, be expanded to 100ml, density is with 1 * 10
6/ ml starts, and detects density and the expression amount of cell every day, and cultured continuously 10 days is recorded cell density and the expression level of the every day of each cell clone, and draws growth curve and express curve.
Result: with traditional screening method comparison, screening method of the present invention adopts containing the fluorescently-labeled expression vector of chimeric ZEO-GFP.The cell clone obtaining through screening, elisa assay result shows, the positive colony rate of screening method of the present invention is 95%, the positive colony rate of conventional screening methods is only 15%, and screening method false positive rate of the present invention is significantly lower than traditional screening method (P < 0.05).
The result of Western Blotting shows (Fig. 2), compare with negative control (pA, pB), transfection the cell of pA-EPO and pB-EPO the expression of target gene can be detected, and in screening method of the present invention (pB-EPO) EPO expression level apparently higher than conventional screening methods (pA-EPO) (P < 0.05).
Through MTX pressurization screening, obtain the cell strain of stably express EPO, cultured continuously detects epo protein expression amount wherein for 10 days afterwards, result shows, screening method of the present invention (expression vector pB-EPO) since the 4th day expression amount obviously than the EPO expression amount high (Fig. 3) in conventional screening methods (expression vector pA-EPO), during by the 10th day, in screening method of the present invention, expressing quantity is 136.9mg/L, and in conventional screening methods, expressing quantity is only 15.5mg/L (table 1, Fig. 3).
Screening method of the present invention adopts containing the fluorescently-labeled expression vector of chimeric ZEO-GFP, particularly chimeric GFP fluorescent mark, can be used flow cell sorter to carry out high-throughput sorting according to fluorescence intensity, has simplified screening step, shorten screening time, thereby greatly improved screening efficiency.
Simultaneously, the present invention expresses in conjunction with utilizing IRES that marker gene DHFR is weakened, when same concentrations MTX pressurizes, the copy number of target genes that expression method of the present invention obtains is far away higher than traditional expression method, thereby the false positive rate in expression system of the present invention screens system well below tradition, and expression level is higher.Therefore conventional screening methods needs MTX pressurization gradient more, at least needs could obtain for 32 weeks the cell strain of stably express.And screening method of the present invention is because its positive rate is high, MTX pressurization gradient is few, the cell strain (table 1) of high expression level target protein that only needed just obtain for 9 weeks.
Each parameter comparison of EPO stable cell line that table 1. conventional screening methods and rapid screening method of the present invention obtain
The above results shows, screening method of the present invention can shorten the time that screens high expression level EPO stable cell line greatly, and significantly improves the expression level of EPO target gene in host cell.
Embodiment 3: the comparison of epo protein expression amount in different hosts cell
Method: the expression plasmid pA-EPO containing target gene EPO that the expression vector pA that embodiment 1 is built and pB and embodiment 2 build and pB-EPO respectively transfection, to following three kinds of host cells, comprise CHO-dhfr cell, bhk cell and HEK293 cell.The screening method of transfection method and acquisition stable cell line is identical with the working method of embodiment 2.
Result: by Western Blotting and elisa assay, the expression of target protein do not detected in the cell of negative control (pA, pB) transfection, therefore fail to screen the stable cell line containing EPO.With negative control comparison, containing the expression plasmid pA-EPO of target gene EPO and the host cell of pB-EPO transfection, through screening, six kinds of stable cell lines have been obtained, elisa assay result shows, in conventional screening methods and screening method of the present invention, epo protein expression amount in CHO host cell is all apparently higher than the expression amount in bhk cell and HEK293 cell, and the epo protein expression amount that the inventive method obtains is apparently higher than traditional method (table 2).
The comparison of table 2. epo protein expression amount (mg/L) in different hosts cell
| Host cell | EPO expression amount in conventional screening methods | EPO expression amount in screening method of the present invention |
| CHO | 15.5 | 136.9 |
| BHK | 9.5 | 87.1 |
| HEK293 | 12.0 | 92.4 |
Embodiment 4: the stable cell line of rapid screening high expression level tPA albumen
According to the method for embodiment 2, build the expression vector that contains target gene tPA, expression plasmid pA-tPA and pB-tPA that acquisition contains tPA, by above-mentioned plasmid and blank plasmid (pA, pB) transfection, to the strain of CHO-dhfr-suspension cell, the line stabilization of going forward side by side is expressed the screening of tPA cell strain.
Result: the cell strain of the expression tPA obtaining through preliminary resistance screening, carry out Western Blotting analysis, result shows: compare with negative control (pA, pB), transfection the cell of pA-tPA and pB-tPA the expression of target gene can be detected, and in screening method of the present invention tPA expression level is apparently higher than conventional screening methods (P < 0.05) (Fig. 4).
The stably express tPA cell strain cultured continuously that pressurization obtains after screening to MTX 10 days, detect the wherein expression amount of tPA albumen, result shows, screening method of the present invention (expression vector pB-tPA) since the 4th day expression amount obviously than the tPA expression amount high (Fig. 5) in conventional screening methods (expression vector pA-tPA), during by the 10th day, in screening method of the present invention, expressing quantity is 157.8mg/L, and in conventional screening methods, expressing quantity is only 30.6mg/L (table 3 and Fig. 5).Conventional screening methods needs MTX pressurization gradient more, at least needs could obtain for 32 weeks the cell strain of stably express, and the screening method MTX of the present invention gradient of pressurizeing is few, the cell strain of high expression level target protein that only needed just obtain for 9 weeks.
In sum, screening method of the present invention can shorten the time that screening obtains high expression level tPA stable cell line greatly, and significantly improves the expression level of tPA target gene in host cell.
Each parameter comparison of tPA stable cell line that table 3 conventional screening methods and rapid screening method of the present invention obtain
Claims (10)
1. a novel mammalian cell expression vector, is characterized in that, described mammalian expression vector only contains single expression cassette, is driven the expression of target gene, selection markers gene and amplification label gene by a strong promoter.Between these genes, by IRES sequence, connect.
2. mammalian cell expression vector according to claim 1, wherein single expression cassette comprises following original paper, and preferably arranges in the following order: (a) strong promoter; (b) target gene cloning site; (c) synthetic IRES element; (d) amplification label gene; (e) FMDV IRES element; (f) synthetic intestinal bacteria EM7 element; (g) chimeric selection markers gene; (h) transcription terminator; (i) be enabled in the initial PUC sequence copying in intestinal bacteria.
3. mammalian cell expression vector according to claim 2, wherein strong promoter comprises CMV, SV40, EF1-α, RSV, MLU, MPSV promotor, preferred CMV promotor, its nucleotide sequence is as shown in SEQ ID NO:1.
4. mammalian cell expression vector according to claim 2, wherein synthetic IRES element comprises FMDV IRES sequence and synthetic GTX IRES sequence, described FMDV IRES nucleotide sequence is as shown in SEQ ID NO:2, and described synthetic GTX IRES nucleotide sequence is as shown in SEQ ID NO:3.
5. mammalian cell expression vector according to claim 2, wherein amplification label gene comprises DHFR gene and GS gene.
6. mammalian cell expression vector according to claim 2, wherein chimeric selection markers gene comprises and has bifunctional antibiotic-screening gene as ZEO, HYP, NEO, with fluorescence protein gene as GFP, the mosaic of RFP, the former can be used for the antibiotic-screening of transformant, and the latter can be used as fluorescent microscope or selected by flow cytometry apoptosis mark.
7. the rapid screening method of the mammalian cell strain of a high expression level foreign protein, the method comprises the following steps: 1) adopt the mammalian cell expression vector described in claim 1-6 any one claim that target gene is transfected into suitable mammalian host cell, 2) utilize the stable cell line of screening method rapid screening high expression level target protein.
8. rapid screening method according to claim 7, wherein said transfection method comprises calcium phosphate method, electricity turns method, liposome transfection, preferred transfection method is that electricity turns method transfection.
9. rapid screening method according to claim 7, wherein said mammalian host cell is selected from Chinese hamster ovary Chinese hamster ovary celI, HEK293, BHK, NS0 and Sp2/0, preferred host cell is Chinese hamster ovary Chinese hamster ovary celI.
10. rapid screening method according to claim 7, it is characterized in that, a plurality of different selection markers genes are combined, adopt chimeric selection markers to carry out the screening of overexpression cell line, described chimeric selection markers comprises the arbitrary combination of the resistant genes such as ZEO, HYP, PUR and GFP, RFP, the combination ZEO-GFP that preferred chimeric selection markers is ZEO and GFP, its nucleotide sequence is as shown in SEQ ID NO:5.
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Cited By (5)
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| CN104630131A (en) * | 2015-01-15 | 2015-05-20 | 上海交通大学 | CHO (Chinese hamster ovary) cell strain for stably expressing human clotting factor VIII and application thereof |
| CN104946687A (en) * | 2015-06-09 | 2015-09-30 | 北京东方百泰生物科技有限公司 | Mammal dual gene highly efficient screening expression vectors and construction method thereof |
| CN105838736A (en) * | 2015-03-24 | 2016-08-10 | 广东东阳光药业有限公司 | Screening method of cell strain of GS expression system |
| CN107338267A (en) * | 2016-05-03 | 2017-11-10 | 中国科学院深圳先进技术研究院 | Two-cistron expression vector and its construction method and application |
| CN110551693A (en) * | 2019-07-22 | 2019-12-10 | 佛山普津生物技术有限公司 | Method for screening HEK cells by antibiotics |
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Cited By (8)
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| CN104630131A (en) * | 2015-01-15 | 2015-05-20 | 上海交通大学 | CHO (Chinese hamster ovary) cell strain for stably expressing human clotting factor VIII and application thereof |
| CN104630131B (en) * | 2015-01-15 | 2017-08-15 | 上海交通大学 | A kind of the Chinese hamster ovary celI strain and its application of the stable factor of expression people blood coagulation eight |
| CN105838736A (en) * | 2015-03-24 | 2016-08-10 | 广东东阳光药业有限公司 | Screening method of cell strain of GS expression system |
| CN105838736B (en) * | 2015-03-24 | 2019-06-25 | 广东东阳光药业有限公司 | A kind of screening technique of GS expression system cell strain |
| CN104946687A (en) * | 2015-06-09 | 2015-09-30 | 北京东方百泰生物科技有限公司 | Mammal dual gene highly efficient screening expression vectors and construction method thereof |
| CN104946687B (en) * | 2015-06-09 | 2018-09-14 | 北京东方百泰生物科技有限公司 | The dual-gene high frequency zone expression vector of mammal and its construction method |
| CN107338267A (en) * | 2016-05-03 | 2017-11-10 | 中国科学院深圳先进技术研究院 | Two-cistron expression vector and its construction method and application |
| CN110551693A (en) * | 2019-07-22 | 2019-12-10 | 佛山普津生物技术有限公司 | Method for screening HEK cells by antibiotics |
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| CN104087612B (en) | 2017-02-22 |
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