CN116445542A - Expression vector and application thereof - Google Patents
Expression vector and application thereof Download PDFInfo
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- CN116445542A CN116445542A CN202210022828.1A CN202210022828A CN116445542A CN 116445542 A CN116445542 A CN 116445542A CN 202210022828 A CN202210022828 A CN 202210022828A CN 116445542 A CN116445542 A CN 116445542A
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- 108090000623 proteins and genes Proteins 0.000 claims abstract description 25
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- 239000003550 marker Substances 0.000 claims description 62
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- 229950010131 puromycin Drugs 0.000 claims description 29
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- 239000013612 plasmid Substances 0.000 claims description 16
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- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 claims description 10
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- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 claims description 8
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- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 claims description 3
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- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
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- SXTAYKAGBXMACB-UHFFFAOYSA-N methionine sulfoximine Chemical compound CS(=N)(=O)CCC(N)C(O)=O SXTAYKAGBXMACB-UHFFFAOYSA-N 0.000 description 2
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- 229940104230 thymidine Drugs 0.000 description 1
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- 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
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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- C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Y105/01003—Dihydrofolate reductase (1.5.1.3)
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Abstract
The invention discloses an expression vector, which can be used for rapidly screening to obtain a cell pool, further screening to obtain a CHO clone to generate a stable CHO cell line, and optimizing a regulatory element in the expression vector to improve the expression quantity of a target protein.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to an expression vector and a method for screening a cell pool by using the same.
Background
Chinese Hamster Ovary (CHO) cells are the most commonly used expression host for the production of complex biomolecules. The two most common methods for expressing proteins in CHO cells are transient gene expression and stable gene expression. Transient gene expression refers to the fact that exogenous genes exist on episomal vectors after entering recipient cells and are not integrated into the chromosomes of the cells. Transient gene expression is the method of choice for rapid production of small amounts of recombinant protein, typically only 1-3 weeks. However, due to the large amount of DNA and host cells required, transient gene expression is not suitable in cases where gram-sized proteins are to be produced. Thus, stable gene expression is typically used to produce gram-sized recombinant proteins. Stable gene expression refers to integration of the transfected gene of interest into chromosomal DNA to express the protein of interest. However, stabilizing gene expression is time consuming and laborious. Typically 3-9 months are required to generate CHO cell lines. Thus, there is a need for a cell line that is rapid, stable, and highly productive of a protein of interest.
Disclosure of Invention
The invention provides an expression vector, which can be used for rapidly screening to obtain a cell pool and further screening to obtain CHO clones, so that a stable CHO cell line is generated. In the invention, the expression quantity of the target protein is improved by optimizing the regulatory element in the expression vector, and the CHO cell line which is rapidly produced is stable and high in yield.
In a first aspect, the invention provides an expression vector comprising a selectable marker expression unit, at least one gene of interest expression unit, and an Inverted Terminal Repeat (ITRs) of a PiggyBac transposon.
The selectable marker expression unit comprises a coding sequence for a selectable marker, and a first regulatory sequence, e.g., a coding sequence for a promoter and a coding sequence for a terminator, operably linked to the coding sequence for the selectable marker.
In some embodiments, the selectable markers include, but are not limited to, puromycin (Puromycin, puro), neomycin (Neomycin, neo), hygromycin B (Hygromycin B, hygrob) and Blasticidin (Blasticidin, bsd), glutamine synthetase (Glutamine synthetase, GS), and dihydrofolate reductase (Dihydrofolate reductase, DHFR). Preferably, the screening marker is human GS.
In some embodiments, the promoter of the selectable marker expression unit may be a herpes simplex virus thymidine kinase (Thymidine kinasegene of Herpes simplex virus, HSV-tk) promoter or a Simian virus40 (SV 40) promoter.
In some embodiments, the terminator of the selectable marker expression unit may be a polyadenylation signal (PolyA), such as SV40 PolyA, bovine growth hormone (Bovine growth hormone, bGH) PolyA, HSV-tk PolyA or Rabbit beta-globin (RBG) PolyA, preferably SV40 PolyA.
Preferably, the selectable marker expression unit comprises a coding sequence for an HSV-tk promoter and a coding sequence for SV40 polyA.
Preferably, the selectable marker expression unit comprises the coding sequence of the HSV-tk promoter, the coding sequence of the human GS selectable marker and the coding sequence of SV40 polyA.
The gene expression unit of interest comprises a gene sequence of interest, and a second regulatory sequence operably linked to the gene sequence of interest, e.g., a coding sequence for a gene expression regulatory element of interest and a coding sequence for a terminator.
In some embodiments, the coding sequence for the gene expression regulatory element of interest has a sequence selected from the group consisting of the sequences set forth in SEQ ID NO. 1-6.
In some embodiments, the terminator of the gene expression unit of interest may be a polyA, such as SV40 polyA, bGH polyA, tk polyA, RBG polyA, preferably bGH polyA.
In some embodiments, the gene of interest is one or more, e.g., 2, 3, or 4, and accordingly, the expression vector has 2, 3, or 4 gene expression units of interest.
In some embodiments, the gene of interest encodes the light chain and/or heavy chain of an anti-CD20 monoclonal antibody (Rituximab) or an anti-HER2 monoclonal antibody (Trastuzumab). Accordingly, the expression vector has 1 or 2 gene expression units of interest.
In some embodiments, the expression vector can be rapidly screened to obtain a CHO cell pool, further screened to obtain a CHO clone to generate a stable CHO cell line, and the expression quantity of the target protein is improved by optimizing a regulatory element in the expression vector, so that the rapidly generated CHO cell line is stable and high in yield.
In a second aspect, the invention provides an expression system comprising the expression vector of the first aspect.
In some embodiments, the expression system further comprises a helper plasmid encoding a PiggyBac transposase.
In a third aspect, the invention provides a host cell transfected with the expression vector of the first aspect.
In some embodiments, the host cell is transfected with the expression system of the second aspect.
Preferably, the host cell has stably integrated into its genome the expression vector of the first aspect.
In some embodiments, the host cell does not comprise a helper plasmid encoding a PiggyBac transposase.
In some embodiments, the host cell is a CHO cell, such as a CHO-K1 cell or a CHOExpressTM cell.
In some embodiments, the host cell is a stable CHO cell line.
In some embodiments, the host cell has increased expression of the protein of interest.
In a fourth aspect, the present invention provides a method of screening cells using the expression vector of the first aspect or the expression system of the second aspect, the method comprising the steps of:
(a) Transfecting a host cell with the expression vector of the first aspect or the expression system of the second aspect; and
(b) A step of screening the host cells obtained in step (a) with a screening agent for a screening marker in the expression vector.
In some embodiments, the host cell is a CHO cell, such as a CHO-K1 cell or a CHOExpressTM cell.
In some embodiments, the selectable marker is puromycin and 10 μg/ml puromycin is used to screen host cells transfected with the expression vector comprising a puromycin resistance selectable marker.
In some embodiments, the selectable marker is puromycin and the host cells transfected with the expression vector comprising the puromycin resistance selectable marker are selected using an increased concentration of puromycin (10 μg/ml, 50 μg/ml, 250 μg/ml).
In some embodiments, the selectable marker is GS and 25 μm methionine iminosulfone (methionine sulfoximine, MSX) is used to screen host cells transfected with the expression vector comprising the GS selectable marker.
In some embodiments, the selectable marker is GS and the host cells transfected with the expression vector comprising the GS selectable marker are screened using an increased concentration of MSX (25 μΜ, 50 μΜ).
In some embodiments, the selectable marker is GS and the host cells transfected with the expression vector comprising the GS selectable marker are screened using an increased concentration of MSX (25 μΜ, 50 μΜ, 100 μΜ).
Preferably, the screening marker is human GS.
In some embodiments, the selectable marker is DHFR and 250nM Methotrexate (MTX) is used to screen host cells transfected with the expression vector comprising the DHFR selectable marker.
In some embodiments, the selectable marker is DHFR and the host cells transfected with the expression vector comprising the DHFR selectable marker are screened using an increased concentration of methotrexate (250 nM, 500 nM).
Preferably, the screening agent is puromycin, methionine iminosulfone or methotrexate.
In some embodiments, the host cells are screened under pressure using CHO cell culture medium containing puromycin, methionine iminosulfone or methotrexate. In some embodiments, the medium is BalanCD CHO GROWTH A medium.
In some embodiments, the screening pressure is removed after the activity has recovered to above 90%.
The screening method provided by the invention screens to obtain the stable CHO clone with high yield, and further generates a stable CHO cell line.
In a fifth aspect, the invention provides cells obtained by the screening method of the fourth aspect.
In a sixth aspect, the present invention provides the use of the expression vector of the first aspect, the expression system of the second aspect, the host cell of the third aspect, or the screening method of the fourth aspect, or the cell of the fifth aspect, for producing a protein of interest with increased expression levels.
The expression vector provided by the invention can be used for rapidly screening to obtain a cell pool, and further screening to obtain a CHO clone to generate a stable CHO cell line, so that the time for generating the stable CHO cell line is greatly shortened, and meanwhile, the expression quantity of target proteins is increased by optimizing regulatory elements in the expression vector, so that the rapidly generated CHO cell line is stable and high in yield.
Description of the drawings:
FIG. 1 shows a block diagram of an expression vector comprising two gene expression units of interest.
FIG. 2 shows a block diagram of an expression vector comprising a gene expression unit of interest.
FIG. 3 shows a screening strategy for cell pools using puromycin resistance screening markers.
Figure 4 shows a screening strategy for cell pools using GS screening markers, including rat GS as well as human GS.
FIG. 5 shows a screening strategy for a cell pool using DHFR screening markers.
FIG. 6 shows the structure of an expression vector for expressing anti-CD20 mAb (Rituximab).
FIG. 7 shows the cell Viability (VIA) of the CHO-K1 cell pool.
FIG. 8 shows CHOExpress TM Cell Viability (VIA) of the cell pool.
FIG. 9 shows the Viable Cell Density (VCD) and the viable cell rate (VIA) of the CHO-K1 cell pool with better recovery of the viable cell rate.
FIG. 10 shows that CHOExpress has better recovery of cell viability TM Cell Density (VCD) and cell Viability (VIA).
FIG. 11 shows a growth curve of a cell pool under 37℃where "KP10" means K1-PR10, "KP250" means K1-PR250, "EP10" means EXP-PR10, "EP250" means EXP-PR250, "KH25" means K1-HG25, "KH50" means K1-HG50, "KH100" means K1-HG100, "EH25" means EXP-HG25, "EH50" means EXP-HG50, "EH100" means EXP-HG100.
FIG. 12 shows the growth curve of the cell pool at a sixth day reduced to 33 ℃, wherein "KP10" means K1-PR10, "KP250" means K1-PR250, "EP10" means EXP-PR10, "EP250" means EXP-PR250, "KH25" means K1-HG25, "KH50" means K1-HG50, "KH100" means K1-HG100, "EH25" means EXP-HG25, "EH50" means EXP-HG50, "EH100" means EXP-HG100.
FIG. 13 shows the results of Protein A HPLC detection of the expression level, wherein, "KP10" means K1-PR10, "KP250" means K1-PR250, "EP10" means EXP-PR10, "EP250" means EXP-PR250, "KH25" means K1-HG25, "KH50" means K1-HG50, "KH100" means K1-HG100, "EH25" means EXP-HG25, "EH50" means EXP-HG50, "EH100" means EXP-HG100.
FIG. 14 shows the growth curve of the cells in example 3.
FIG. 15 shows the results of the measurement of the expression level in example 3.
FIG. 16 shows the results of the expression level detection in example 4.
Detailed Description
In some embodiments, the invention may construct an expression vector comprising two gene expression units of interest (see FIG. 1), or an expression vector comprising one gene expression unit of interest (see FIG. 2).
The PiggyBac system adopted by the invention comprises two vectors, wherein one vector is called auxiliary plasmid and is responsible for encoding transposase; another vector is called a transposon plasmid, which contains two terminal repeats (ITRs) and a transposed region between them, to which the promoter, the expression cassette and the gene of interest are cloned together. When the helper plasmid and transposon plasmid co-transfect target cells, the transposase produced by the helper plasmid will recognize both ITR elements of the transposon, and then the transposed region and both ITR elements will be inserted into the host genome. The transposon insertion usually occurs at a host chromosomal site comprising TTAA sequences and TTAA repeats occur on both sides of the transposon.
PiggyBac belongs to class II transposons, which move by a "cut-and-paste" mechanism, transposing from one place to another without leaving the sequence itself. The helper plasmid is gradually lost due to its transient transfection into the host cell. With the loss of helper plasmid, the transposon becomes permanently integrated in the host genome. When these host cells are transfected again with helper plasmids, the integrated transposon will again move through a "cut-and-paste" mechanism.
In the method of screening a cell pool using the expression vector of the present invention or the expression system of the present invention,
the screening strategy for cell pools using puromycin resistance screening markers is as follows (see fig. 3):
puro strategy 1: screening was performed using 10. Mu.g/ml puromycin throughout the course;
puro strategy 2: screening was performed using an increased concentration of puromycin (10. Mu.g/ml, 50. Mu.g/ml, 250. Mu.g/ml);
the screening strategy for cell pools using GS screening markers, including rat GS and human GS, is as follows (see fig. 4):
GS strategy 1: screening was performed using 25 μm MSX throughout the course;
GS strategy 2: screening was performed using varying concentrations of MSX (25. Mu.M, 50. Mu.M);
GS strategy 3: screening was performed using varying concentrations of MSX (25. Mu.M, 50. Mu.M, 100. Mu.M);
the screening strategy for cell pools using DHFR screening markers is as follows (see fig. 5):
DHFR strategy 1: screening was performed using 250nM Methotrexate (MTX);
DHFR strategy 2: the screening was performed using MTX (250 nM, 500 nM) with varying concentration.
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way.
Example 1: construction of expression vector for expressing anti-CD20 mAb (Rituximab) and screening of cell lines
Construction and screening of CHO cell lines
Referring to FIG. 6, the construction of an expression vector for expressing anti-CD20 mAb (Rituximab) is shown.
The coding sequence of the target gene expression regulatory element is shown as SEQ ID NO.1, the LTD sequence of the PiggyBac transposon is shown as SEQ ID NO.7, the RTD sequence of the PiggyBac transposon is shown as SEQ ID NO.8, the bGH polyA sequence is shown as SEQ ID NO.9, the HSV-tk promoter sequence is shown as SEQ ID NO.10, and the SV40 polyA sequence is shown as SEQ ID NO. 11.
Transfection system:
PEI (25 KD) 37.5 μg; 11.25 μg of recombinant anti-CD20 monoclonal antibody plasmid (see expression vector for expressing anti-CD20 mAb (Rituximab) constructed in FIG. 6); 1.25 μg of transposase plasmid; hyCell Tra containing CHO cells1.5ml of nsfx-C medium (GE, SH 30934.04); density of CHO cells in culture medium 15×10 6 cell/ml。
The transfection method comprises the following steps:
during transfection, PEI, recombinant anti-CD20 monoclonal antibody plasmid, transposase plasmid and Hycell Transfx-C culture medium containing CHO cells are evenly mixed and placed in a shaking table at 37 ℃ for incubation, and 3.5ml BalanCD CHO GROWTH A is added after 1 hour of transfection.
The screening method comprises the following steps:
two days after transfection, pressure screening was performed with BalanCD CHO GROWTH A medium containing screening reagent, and passaged every three days at a passaged cell density of 0.3-0.5X10 6 And (5) removing the screening pressure after the cell/ml is recovered to the activity rate of more than 90%.
CHO-K1 (available from ATCC) and CHOExpress are used in this example TM Two host cells (purchased from excelgene), and four screening markers puromycin, rat GS, human GS, and mouse DHFR, constructed eight cells expressing anti-CD20 monoclonal antibodies, and different cell pool screening strategies were employed for these cells, as detailed in table 1 below.
Table 1:
| group of | Screening markers | Cell lines | Screening strategies |
| K1-PR10 | Puromycin resistance selectable marker | CHO-K1 | Puro strategy 1 |
| K1-PR250 | Puromycin resistance selectable marker | CHO-K1 | Puro strategy 2 |
| K1-RG25 | Rat GS | CHO-K1 | GS strategy 1 |
| K1-RG50 | Rat GS | CHO-K1 | GS strategy 2 |
| K1-RG100 | Rat GS | CHO-K1 | GS strategy 3 |
| K1-HG25 | Human GS | CHO-K1 | GS strategy 1 |
| K1-HG50 | Human GS | CHO-K1 | GS strategy 2 |
| K1-HG100 | Human GS | CHO-K1 | GS strategy 3 |
| K1-MD250 | Mouse DHFR | CHO-K1 | DHFR strategy 1 |
| K1-MD500 | Mouse DHFR | CHO-K1 | DHFR strategy 2 |
| EXP-PR10 | Puromycin resistance selectable marker | CHOExpress TM | Puro strategy 1 |
| EXP-PR250 | Puromycin resistance selectable marker | CHOExpress TM | Puro strategy 2 |
| EXP-RG25 | Rat GS | CHOExpress TM | GS strategy 1 |
| EXP-RG50 | Rat GS | CHOExpress TM | GS strategy 2 |
| EXP-RG100 | Rat GS | CHOExpress TM | GS strategy 3 |
| EXP-HG25 | Human GS | CHOExpress TM | GS strategy 1 |
| EXP-HG50 | Human GS | CHOExpress TM | GS strategy 2 |
| EXP-HG100 | Human GS | CHOExpress TM | GS strategy 3 |
| EXP-MD250 | Mouse DHFR | CHOExpress TM | DHFR strategy 1 |
| EXP-MD500 | Mouse DHFR | CHOExpress TM | DHFR strategy 2 |
The cell Viability (VIA) of CHO-K1 cell pools is shown in fig. 7 and table 2 below.
Table 2:
from the above results, it was found that the cell pool using puromycin resistance marker and human GS marker was recovered within two weeks after transfection, and that the cell pool using DHFR marker was recovered much slower. In addition, the concentration of the screening agent during the screening process has a certain effect on the recovery of the cell pool.
CHOExpress TM Cell viability of the cell pool is shown in fig. 8 and table 3 below:
table 3:
| Day 11 | Day 14 | Day 17 | |
| EXP-HG25 | 95.4 | 97.67 | 97.86 |
| EXP-HG50 | 97.12 | 95.84 | 97.28 |
| EXP-HG100 | 97.12 | 91.82 | 92.11 |
| EXP-MD250 | 24.58 | 36.99 | 35.82 |
| EXP-MD500 | 24.14 | 24.63 | 62.62 |
| EXP-PR10 | 98.59 | 99.28 | 99.24 |
| EXP-PR250 | 77.86 | 95.82 | 89.54 |
| EXP-RG25 | 68.91 | 79.43 | 84.11 |
| EXP-RG50 | 54.76 | 60.68 | 75.38 |
| EXP-RG100 | 54.76 | 59.89 | 63.19 |
cell viability recovery the Viable Cell Density (VCD) and cell Viability (VIA) of the CHO-K1 cell pool was better recovered (FIG. 9).
CHOExpress with better cell viability recovery TM Cell viability density (VCD) and cell Viability (VIA) were measured (see fig. 10).
Example 2: feed batch culture evaluation cell pools using human GS and puromycin resistance selection markers
The cells after the screening were treated at 0.5X10 6 cell density of cell/ml (seed by centrifuge) was inoculated into 50ml of a medium containing 0.2% of an anti-caking agent (ACA) EX-CELL Advanced CHO Fed-batch, and the mixture was subjected to shaking culture after mixing. The culture conditions are as follows: 37 ℃,140rpm,5% CO 2 85% humidity.
And (3) a feeding strategy:
3%BalanCD CHO Feed 4+0.3%Cell Boost 7b on days 2, 4, 6, 8, 10 of culture;
when the glucose concentration is lower than 4g/L, adding glucose to the concentration of 8g/L;
for the cell pool using puromycin resistance selection marker, glutamine concentration was maintained at not less than 4mM on days 2-5 of culture.
A set of conditions was also set up to adjust the temperature to 33℃on day 6 of incubation, with the other conditions being the same as described above.
The growth curve of the cell pool at 37℃is shown in FIG. 11.
The growth curve of the cell pool at the sixth day reduced to 33℃is shown in FIG. 12.
As can be seen from the growth curve of FIG. 12, the trend of the cells grown at the temperature of 33℃was similar to that of the cells kept at 37℃in the sixth day, but the cells survived longer when the temperature was lowered to 33 ℃.
The results of Protein A HPLC detection of the expression level are shown in FIG. 13.
Example 3: monoclonalization of positive clone cells and detection of expression level of monoclonal cell strain
And selecting the monoclonal from the cell pool with the highest expression level by a limiting dilution method, and finally, aiming at each monoclonal, carrying out amplification culture, and detecting the expression level of the target protein in the culture supernatant by an Octet molecular interaction instrument to obtain the high-yield monoclonal cell strain.
Taking the positive monoclonal Cell strain screened in the step, and carrying out Fed batch culture on the positive monoclonal Cell strain by taking Advanced CHO Fed-batch as a basic culture medium and taking Cell Boost 7a/7b as a feed culture medium. In the evaluation of cell expression level, cells are inoculated into 250ml shake flasks at a density of 0.3 x 10≡6cells/ml, and the culture volume is 50ml,140rpm,5% CO 2 Culturing at 37 ℃.
And (3) a feeding strategy:
cells were replenished 3%Cell Boost 7a+0.3%Cell Boost 7b daily from day 3.
When the glucose concentration was less than 5g/L, glucose was added to a concentration of 8g/L.
The cell growth curve and the expression level are shown in FIGS. 14 and 15, respectively.
Example 4
The expression vector of the anti-HER2 monoclonal antibody (Trastuzumab) is constructed by adopting the expression vector in the embodiment of the invention, and a monoclonal cell strain is obtained through transfection (CHO-K1 is used as a host cell), cell pool screening (GS strategy 3) and monoclonal of positive clone cells. And taking the screened positive monoclonal Cell strain, and carrying out fed-batch culture on the positive monoclonal Cell strain by taking HyClone Actipro Cell culture medium as a basic culture medium and taking Cell Boost 7a/7b as a feed culture medium. In the evaluation of cell expression level, cells were inoculated at a density of 10X 10-6 cells/ml in 250ml shake flasks at a culture volume of 50ml,140rpm,5% CO 2 Culturing at 37 ℃.
And (3) a feeding strategy:
initially add 2% Cell Boost 7a and 2% Cell Boost 7b;
when the VCD of the Cell is more than 20 x 10 x 6cells/ml, adding 4% of Cell Boost 7a and 4% of Cell Boost 7b;
when the VCD of the Cell is more than 30 x 10 x 6cells/ml, 3% of Cell Boost 7a and 0.6% of Cell Boost 7b are added;
when the glucose concentration was less than 5g/L, glucose was added to a concentration of 8g/L.
The expression level is shown in FIG. 16.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
In addition, any combination of the various embodiments of the present invention may be performed, so long as the concept of the present invention is not violated, and the technical solution of the present invention should also be considered as being not limited to the above specific examples, and all technical modifications made according to the technical solution of the present invention fall within the protection scope of the present invention.
Sequence listing
<110> Momordica Charantia Biotech Co Ltd
CANTONBIO Co.,Ltd.
Foshan Pu Jin Bioisystech Co.,Ltd.
<120> expression vector and use thereof
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tggaatttcc aggggaaaac cggggcggtg ttacgttttg gctgcccttt cacttcccat 180
tgacgtgtat tggctcgaga acggtacttt cccattaatc agctatggga aagtaccgtt 240
taaaggtcac gttgcattag tttcaatagc ccattgacgt caatggtggg aaagtacatg 300
gcgttttaat taaattggct ggaaaaaccc aatgactcac ccctattgac cttatgtacg 360
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<213> Artificial Sequence
<400> 4
gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240
atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420
agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480
tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540
aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcct gaaatggaag 600
aaaaaaactt tgaaccactg tctgaggctt gagaatgaac caagatccaa actcaaaaag 660
ggcaaattcc aaggagaatt acatcaagtg ccaagctggc ctaacttcag tctccaccca 720
ctcagtgtgg ggaaactcca tcgcataaaa cccctccccc caacctaaag acgacgtact 780
ccaaaagctc gagaactaat cgaggtgcct ggacggcgcc cggtactccg tggagtcaca 840
tgaagcgacg gctgaggacg gaaaggccct tttcctttgt gtgggtgact cacccgcccg 900
ctctcccgag cgccgcgtcc tccattttga gctccctgca gcagggccgg gaagcggcca 960
tctttccgct cacgcaactg gtgccgaccg ggccagcctt gccgcccagg gcggggcgat 1020
acacggcggc gcgaggccag gcaccagagc aggccggcca gcttgagact acccccgtcc 1080
gattctcggt ggccgcgctc gcaggccccg cctcgccgaa catgtgcgct gggacgcacg 1140
ggccccgtcg ccgcccgcgg ccccaaaaac cgaaatacca gtgtgcagat cttggcccgc 1200
atttacaaga ctatcttgcc agaaaaaaag cgtcgcagca ggtcatcaaa aattttaaat 1260
ggctagagac ttatcgaaag cagcgagaca ggcgcgaagg tgccaccaga ttcgcacgcg 1320
gcggccccag cgcccaggcc aggcctcaac tcaagcacga ggcgaagggg ctccttaagc 1380
gcaaggcctc gaactctccc acccacttcc aacccgaagc tcgggatcaa gaatcacgta 1440
ctgcagccag gggcgtggaa gtaattcaag gcacgcaagg gccataaccc gtaaagaggc 1500
caggcccgcg ggaaccacac acggcactta c 1531
<210> 5
<211> 2122
<212> DNA/RNA
<213> Artificial Sequence
<400> 5
ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60
ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120
gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180
gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240
gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300
acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 360
gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 420
cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 480
ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 540
caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 600
gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 660
gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 720
ggtctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 780
gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 840
acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 900
tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 960
tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg 1020
gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa 1080
ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1140
gtggttcaaa gtttttttct tccatttcag gtgtcgtgac tgaaatggaa gaaaaaaact 1200
ttgaaccact gtctgaggct tgagaatgaa ccaagatcca aactcaaaaa gggcaaattc 1260
caaggagaat tacatcaagt gccaagctgg cctaacttca gtctccaccc actcagtgtg 1320
gggaaactcc atcgcataaa acccctcccc ccaacctaaa gacgacgtac tccaaaagct 1380
cgagaactaa tcgaggtgcc tggacggcgc ccggtactcc gtggagtcac atgaagcgac 1440
ggctgaggac ggaaaggccc ttttcctttg tgtgggtgac tcacccgccc gctctcccga 1500
gcgccgcgtc ctccattttg agctccctgc agcagggccg ggaagcggcc atctttccgc 1560
tcacgcaact ggtgccgacc gggccagcct tgccgcccag ggcggggcga tacacggcgg 1620
cgcgaggcca ggcaccagag caggccggcc agcttgagac tacccccgtc cgattctcgg 1680
tggccgcgct cgcaggcccc gcctcgccga acatgtgcgc tgggacgcac gggccccgtc 1740
gccgcccgcg gccccaaaaa ccgaaatacc agtgtgcaga tcttggcccg catttacaag 1800
actatcttgc cagaaaaaaa gcgtcgcagc aggtcatcaa aaattttaaa tggctagaga 1860
cttatcgaaa gcagcgagac aggcgcgaag gtgccaccag attcgcacgc ggcggcccca 1920
gcgcccaggc caggcctcaa ctcaagcacg aggcgaaggg gctccttaag cgcaaggcct 1980
cgaactctcc cacccacttc caacccgaag ctcgggatca agaatcacgt actgcagcca 2040
ggggcgtgga agtaattcaa ggcacgcaag ggccataacc cgtaaagagg ccaggcccgc 2100
gggaaccaca cacggcactt ac 2122
<210> 6
<211> 2676
<212> DNA/RNA
<213> Artificial Sequence
<400> 6
ctcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240
atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca 420
tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag 480
cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc 540
ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt 600
ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg 660
cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc 720
cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc 780
ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg 840
aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt 900
gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc 960
gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg 1020
ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg 1080
tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc 1140
acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc 1200
gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg 1260
cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg 1320
gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg 1380
cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca 1440
ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg 1500
agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc 1560
cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 1620
tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc 1680
tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgctgaaat 1740
ggaagaaaaa aactttgaac cactgtctga ggcttgagaa tgaaccaaga tccaaactca 1800
aaaagggcaa attccaagga gaattacatc aagtgccaag ctggcctaac ttcagtctcc 1860
acccactcag tgtggggaaa ctccatcgca taaaacccct ccccccaacc taaagacgac 1920
gtactccaaa agctcgagaa ctaatcgagg tgcctggacg gcgcccggta ctccgtggag 1980
tcacatgaag cgacggctga ggacggaaag gcccttttcc tttgtgtggg tgactcaccc 2040
gcccgctctc ccgagcgccg cgtcctccat tttgagctcc ctgcagcagg gccgggaagc 2100
ggccatcttt ccgctcacgc aactggtgcc gaccgggcca gccttgccgc ccagggcggg 2160
gcgatacacg gcggcgcgag gccaggcacc agagcaggcc ggccagcttg agactacccc 2220
cgtccgattc tcggtggccg cgctcgcagg ccccgcctcg ccgaacatgt gcgctgggac 2280
gcacgggccc cgtcgccgcc cgcggcccca aaaaccgaaa taccagtgtg cagatcttgg 2340
cccgcattta caagactatc ttgccagaaa aaaagcgtcg cagcaggtca tcaaaaattt 2400
taaatggcta gagacttatc gaaagcagcg agacaggcgc gaaggtgcca ccagattcgc 2460
acgcggcggc cccagcgccc aggccaggcc tcaactcaag cacgaggcga aggggctcct 2520
taagcgcaag gcctcgaact ctcccaccca cttccaaccc gaagctcggg atcaagaatc 2580
acgtactgca gccaggggcg tggaagtaat tcaaggcacg caagggccat aacccgtaaa 2640
gaggccaggc ccgcgggaac cacacacggc acttac 2676
<210> 7
<211> 309
<212> DNA/RNA
<213> Artificial Sequence
<400> 7
gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata 60
acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca 120
ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac 180
gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg 240
ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc 300
tttctaggg 309
<210> 8
<211> 238
<212> DNA/RNA
<213> Artificial Sequence
<400> 8
ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc 60
atgtgtttta tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt 120
tattatattt acacttacat actaataata aattcaacaa acaatttatt tatgtttatt 180
tatttattaa aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aactttta 238
<210> 9
<211> 225
<212> DNA/RNA
<213> Artificial Sequence
<400> 9
ccatagagcc caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc 60
tgtcctgccc caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa 120
tttcctcatt ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca 180
cgggggaggg gcaaacaaca gatggctggc aactagaagg cacag 225
<210> 10
<211> 203
<212> DNA/RNA
<213> Artificial Sequence
<400> 10
cttcatcccc gtggcccgtt gctcgcgttt gctggcggtg tccccggaag aaatatattt 60
gcatgtcttt agttctatga tgacacaaac cccgcccagc gtcttgtcat tggcgaaaac 120
acgcagatgc agtcggggcg gcgcggtccc aggtccactt cgcatattaa ggtgacgcgt 180
gtggcctcga acacagagcg act 203
<210> 11
<211> 135
<212> DNA/RNA
<213> Artificial Sequence
<400> 11
gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga 60
aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc 120
tgcaataaac aagtt 135
Claims (12)
1. An expression vector comprising a selectable marker expression unit, at least one gene of interest expression unit, and an inverted terminal repeat of a PiggyBac transposon.
2. Expression vector according to claim 1, wherein the selectable marker expression unit comprises a coding sequence for a selectable marker and a first regulatory sequence operably linked to the coding sequence for the selectable marker, preferably comprising a coding sequence for a promoter and a coding sequence for a terminator.
3. The expression vector according to claim 2, wherein the selection marker is puromycin, neomycin, hygromycin B, and blasticidin, glutamine synthetase, or dihydrofolate reductase, preferably the selection marker is human glutamine synthetase.
4. The expression vector according to claim 2, wherein the promoter of the selectable marker expression unit is a herpes simplex virus thymidine kinase promoter, a simian virus40 promoter, and/or
The terminator of the screening marker expression unit is a simian vacuole virus40 polyadenylation signal, a bovine growth hormone polyadenylation signal, a herpes simplex virus thymidine kinase polyadenylation signal or a rabbit beta-globin polyadenylation signal, preferably a simian vacuole virus40 polyadenylation signal;
preferably, the selectable marker expression unit includes a coding sequence for a herpes simplex virus thymidine kinase promoter and a coding sequence for a simian vacuolated virus40 polyadenylation signal;
preferably, the selectable marker expression unit includes a coding sequence for a herpes simplex virus thymidine kinase promoter, a coding sequence for a human glutamine synthetase selectable marker, and a coding sequence for a monkey vacuolated virus40 polyadenylation signal.
5. The expression vector of claim 1, wherein the gene expression unit of interest comprises a gene sequence of interest and a second regulatory sequence operably linked to the gene sequence of interest,
preferably, the second regulatory sequence comprises a coding sequence for a gene expression regulatory element of interest and a coding sequence for a terminator;
preferably, the coding sequence of the gene expression regulatory element of interest has a sequence selected from the group consisting of the sequences shown in SEQ ID NO. 1-6;
preferably, the terminator of the target gene expression unit is a simian vacuolated virus40 polyadenylation signal, a bovine growth hormone polyadenylation signal, a herpes simplex virus thymidine kinase polyadenylation signal or a rabbit β -globin polyadenylation signal, preferably a bovine growth hormone polyadenylation signal.
6. An expression system comprising the expression vector of any one of claims 1-5.
7. The expression system according to claim 6, further comprising a helper plasmid encoding a PiggyBac transposase.
8. A host cell transfected with the expression vector according to claim 1 to 5, or the expression system according to claim 6 or 7,
preferably, the host cell has stably integrated into its genome the expression vector of any one of claims 1-5;
preferably, the host cell does not comprise a helper plasmid encoding a PiggyBac transposase;
preferably, the host cell is a CHO cell.
9. A method of screening cells using the expression vector of any one of claims 1 to 5 or the expression system of claim 6 or 7, said method comprising the steps of:
(a) Transfecting a host cell with the expression vector of any one of claims 1 to 5 or the expression system of claim 6 or 7; and
(b) A step of screening the host cells obtained in step (a) with a screening agent for a screening marker in the expression vector.
10. The method of claim 9, wherein the host cell is a CHO cell;
preferably, the selectable marker is puromycin and 10 μg/ml puromycin is used to screen host cells transfected with the expression vector comprising a puromycin resistance selectable marker;
preferably, the selectable marker is puromycin and the host cells transfected with the expression vector comprising a puromycin resistance selectable marker are selected using an increased concentration of puromycin of 10 μg/ml, 50 μg/ml, 250 μg/ml;
preferably, the selectable marker is glutamine synthetase and 25 μm methionine iminosulfone is used to screen host cells transfected with the expression vector comprising a glutamine synthetase selectable marker;
preferably, the selectable marker is glutamine synthetase and the host cells transfected with the expression vector comprising a glutamine synthetase selectable marker are selected using 25 μm, 50 μm elevated methionine iminosulfone;
preferably, the selectable marker is a glutamine synthetase selectable marker and the host cells transfected with the expression vector of the glutamine synthetase selectable marker are selected using 25 μm, 50 μm, 100 μm elevated methionine iminosulfone;
preferably, the screening marker is human glutamine synthetase;
preferably, the selectable marker is dihydrofolate reductase and 250nM methotrexate is used to screen host cells transfected with the expression vector comprising a dihydrofolate reductase selectable marker;
preferably, the selectable marker is dihydrofolate reductase and the host cells transfected with the expression vector comprising the dihydrofolate reductase selectable marker are screened using methotrexate at an increased concentration of 250nM, 500 nM.
11. A cell obtained by the screening method of claim 10.
12. Use of the expression vector of any one of claims 1 to 5, the expression system of claim 6 or 7, the host cell of claim 8, or the screening method of claim 9 or 10, or the cell of claim 11 for the production of a protein of interest with increased expression.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210022828.1A CN116445542A (en) | 2022-01-10 | 2022-01-10 | Expression vector and application thereof |
| PCT/CN2023/071371 WO2023131330A1 (en) | 2022-01-10 | 2023-01-09 | Expression vector and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210022828.1A CN116445542A (en) | 2022-01-10 | 2022-01-10 | Expression vector and application thereof |
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| Publication Number | Publication Date |
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| CN116445542A true CN116445542A (en) | 2023-07-18 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210022828.1A Pending CN116445542A (en) | 2022-01-10 | 2022-01-10 | Expression vector and application thereof |
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| CN (1) | CN116445542A (en) |
| WO (1) | WO2023131330A1 (en) |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090042297A1 (en) * | 2007-06-01 | 2009-02-12 | George Jr Alfred L | Piggybac transposon-based vectors and methods of nucleic acid integration |
| CN106755096A (en) * | 2016-12-20 | 2017-05-31 | 上海药明生物技术有限公司 | The method for obtaining the stable cell mass of expression target protein in Chinese hamster ovary celI using piggyBac transposon |
| WO2020068631A1 (en) * | 2018-09-24 | 2020-04-02 | Merck Sharp & Dohme Corp. | Expression vectors for eukaryotic expression systems |
| EP3898965A4 (en) * | 2018-12-21 | 2022-10-12 | Merck Sharp & Dohme Corp. | Expression vectors for eukaryotic expression systems |
| WO2020210239A1 (en) * | 2019-04-08 | 2020-10-15 | Dna Twopointo Inc. | Integration of nucleic acid constructs into eukaryotic cells with a transposase from oryzias |
| IL301621A (en) * | 2019-04-08 | 2023-05-01 | Dna Twopointo Inc | Transposition of nucleic acid constructs into eukaryotic genomes with a transposase from amyelois |
| EP4107272A2 (en) * | 2020-02-19 | 2022-12-28 | Wuxi Biologics Ireland Limited | Enhanced expression system and methods of use thereof |
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| WO2023131330A1 (en) | 2023-07-13 |
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