CN113025648A - Method for transiently expressing target protein in Expi293 cell and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biology, and particularly provides a method for expressing a target protein transiently by an Expi293 cell and application thereof. According to the method for transiently expressing the target protein by the Expi293 cell, the step of culturing the Expi293 cell transfected with the target DNA by using the culture medium comprises cooling treatment. By adopting the operation, the expression quantity and the activity of the target protein can be greatly improved, and the defects of low transfection efficiency and low expression quantity in the prior art are overcome.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for expressing a target protein transiently by an Expi293 cell and application thereof.
Background
The production of recombinant proteins in milligram or gram grades is a fundamental process in preclinical, biochemical, biophysical and pharmaceutical research in many research teams and pharmaceutical companies. Currently, there are a number of expression systems that can produce recombinant proteins, including E.coli, eukaryotic yeast, baculovirus-mediated insect cell, and mammalian expression systems. Coli expression systems are simple to operate, low in culture cost, and capable of rapidly producing high levels of recombinant proteins, but suffer from poor recombinant protein solubility or lack of appropriate post-translational modifications. Eukaryotic yeast expression systems, particularly Pichia pastoris and Saccharomyces cerevisiae, offer some capabilities for substitution and post-translational modification of proteins in prokaryotic hosts (e.g., E.coli hosts), however, yeast expression systems are not popular in many laboratories due to technical requirements. Baculovirus-insect cell expression systems have eukaryotic protein synthesis capacity and provide protein folding, modification and processing similar to that observed in mammalian cells, and then the major drawback of baculovirus-mediated insect cell expression systems is that it takes a lot of time and effort to express the target protein, and the protein N-glycosylation pathway of insect cells is unable to produce complex dibasic N-linked oligosaccharide side chains with penultimate galactose and terminal sialic acid residues. In contrast to the three systems described above, the mammalian cell system for the production of recombinant proteins is the best choice for the biopharmaceutical field for its function of optimally expressing high molecular weight protein products, producing the correct folding of proteins of complex structure and providing appropriate post-translational modifications to glycoproteins.
The traditional mammalian cell expression system is constructed as a stable strain expression system which is stably expressed by cells and integrates exogenous genes on the chromosome of a host cell. Construction of a stable strain expression system for recombinant protein production requires a long screening after introduction of DNA into cells before a highly expressed and stable cell strain can be identified, which usually takes several months and takes a lot of manpower to complete.
With the rapid development of life sciences and biopharmaceuticals, a large number of proteins are used for basic research and drug development, and a method for obtaining recombinant proteins in a short time is urgently needed. Transient expression of genes arises, and unlike expression systems of stable strains, transiently transfected foreign DNA is not integrated into host cell DNA, and the number thereof in cells generally decreases with cell division, so that protein expression can be maintained for only several days to more than ten days, but has advantages of availability of expression products of genes within a few days, and strong versatility. However, compared with the construction of a stable strain expression system, transient transfection has the disadvantages of low transfection efficiency and low expression amount, so that the application of the method is greatly limited, and the cost of transient transfection is high although transfection reagents, culture methods and the like are gradually mature. Therefore, how to improve the efficiency of transient transfection, improve the expression level and reduce the cost of transient transfection becomes a problem to be solved urgently in daily operation.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first objective of the present invention is to provide a method for transiently expressing a target protein in Expi293 cells, so as to alleviate at least one of the above technical problems, including the problem of excessive cost for transiently transfecting Expi293 cells to express the target protein in the prior art.
The second purpose of the invention is to provide the application of the method in immunoassay.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method for transiently expressing a target protein in Expi293 cells comprises the step of culturing the Expi293 cells transfected with target DNA, wherein the culturing step comprises cooling treatment.
Further, the step of culturing the Expi293 cells after transfection with the target DNA comprises: culturing at 36-38 deg.C, cooling to 32-34 deg.C for 1-3 days, and culturing for 4-10 days.
Further, the culturing step further comprises a medium replacement resuspension process;
preferably, the step of culturing the Expi293 cells after transfection with the target DNA comprises: culturing at 36-38 deg.C, cooling to 30-34 deg.C for 1-3 days, culturing, changing culture medium for 3-5 days, and performing heavy suspension culture for 6-10 days;
preferably, the step of culturing the Expi293 cells after transfection with the target DNA comprises: culturing at 37 deg.C, cooling to 33 deg.C for culturing on day 2, changing culture medium on day 4, and performing resuspension culture until day 6.
Further, the target DNA is transfected into the Expi293 cell by a transfection reagent to obtain the Expi293 cell after the target DNA is transfected;
preferably, the ratio of the target DNA to the Expi293 cells is (15-30) μ g: (2X 10)7-5×107) And (4) respectively.
Further, the mass ratio of the target DNA to the transfection reagent is 1: (2-4).
Further, the mass ratio of the target DNA to the transfection reagent is 1: 3.
further, the transfection reagent comprises PEI or liposomes.
Further, the transfection reagent is PEI.
Further, the medium includes Dynamis medium.
The use of the above method for recombinant protein expression.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for transiently expressing a target protein by an Expi293 cell, which comprises the step of culturing the Expi293 cell transfected with a target DNA, wherein the step of culturing comprises cooling treatment. The inventor discovers for the first time that the difference of cell culture process parameters can influence the expression quantity and activity of target protein, researches show that the temperature reduction operation in the culture process can greatly influence the expression quantity and activity of the target protein transiently expressed by Expi293 cells, and the operation can greatly improve the expression quantity and activity of the target protein, thereby overcoming the defects of low transfection efficiency and low expression quantity in the prior art. The method is suitable for industrial production, and is more favorable for the normal operation of the instantaneous transfection and expression of target protein by the Expi293 cell.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
A method for transiently expressing a target protein in Expi293 cells comprises the step of culturing the Expi293 cells transfected with target DNA, wherein the culturing step comprises cooling treatment.
The inventor discovers for the first time that the difference of cell culture process parameters can influence the expression quantity and activity of target protein, and researches show that the temperature reduction operation in the culture process can greatly influence the expression quantity and activity of the target protein transiently expressed by Expi293 cells. The method is suitable for industrial production, and is more favorable for the normal operation of the instantaneous transfection and expression of target protein by the Expi293 cell.
The temperature reduction treatment can be that the culture is carried out after the conventional culture is carried out for a period of time and then the culture temperature is reduced; the conventional culture in the present invention refers to the general procedure of Expi293 cells, i.e., continuous culture at 37 ℃.
In a preferred embodiment, the step of culturing Expi293 cells after transfection with the DNA of interest comprises: culturing at 36-38 deg.C, cooling to 32-34 deg.C for 1-3 days, and culturing for 4-10 days. The inventor discovers that the culture temperature influences the expression quantity and the activity of the target protein through research, and discovers that the expression quantity and the activity of the target protein can be further improved by continuously culturing at 36-38 ℃ and then culturing at the temperature of 32-34 ℃ on days 1-3. The temperature of the temperature reduction culture can be, but is not limited to, 32, 33 or 34 ℃; the time of the temperature-decreasing culture may be, but is not limited to, day 1, day 1.5, day 2, day 2.5 or day 3 of the continuous culture.
In a preferred embodiment, the culturing step further comprises a replacement medium resuspension process. The replacement medium resuspension treatment refers to replacing the cell culture medium in the conventional culture process of the Expi293 cells transfected with the target DNA, continuously culturing the resuspended cells, and replacing the replaced culture medium with a new unused culture medium. The inventors found through research that re-suspending cells with new culture medium on days 3-5 of cell culture and continuing the culture can further improve the expression and activity of the target protein, and the time for changing the culture medium can be, but is not limited to, day 3, day 3.5, day 4, day 4.5 or day 5 of continuous culture.
In a preferred embodiment, the step of culturing Expi293 cells after transfection with the DNA of interest comprises: culturing at 36-38 deg.C, cooling to 30-34 deg.C for 1-3 days, culturing, changing culture medium for 3-5 days, and performing resuspension culture for 6-10 days. The inventor researches and finds that the combination of the operations of temperature reduction and culture medium replacement based suspension culture can further improve the expression amount and activity of the target protein and obtain better technical effect. For example, the culture is carried out at 37 ℃ first, the temperature is reduced to 33 ℃ on day 2, the medium is replaced on day 4, and the culture is resuspended on day 6.
In a preferred embodiment, the target DNA is transfected into Expi293 cells by a transfection reagent, resulting in Expi293 cells after transfection of the target DNA.
In a preferred embodiment, the ratio of target DNA to Expi293 cells is (15-30) μ g: (2X 10)7-5×107) And (4) respectively. Compared with target DNA, the Expi293 cells are too many or too few, the transient transfection efficiency is too low, the cells capable of expressing target protein are too few or waste of the target DNA is caused, and the cost is higher. The inventor finds that the ratio of the DNA to the Expi293 cell has a certain preferable range, and the transient transfection efficiency is higher in the range provided by the invention. Expi293 cells can be, but are not limited to, 2X 10 when the target DNA is typically, but not limited to, 15. mu.g, 17. mu.g, 20. mu.g, 23. mu.g, 25. mu.g, 27. mu.g, or 30. mu.g72.5 x 1072, 3 x 1073.5 x 1074 x 10 pieces74.5 x 107Or 5X 107And (4) respectively.
In a preferred embodiment, the mass ratio of target DNA to transfection reagent is 1: (2-4). The inventors have optimized and defined the ratio of target DNA to transfection reagent, in a mass ratio of 1: (2-4), the transfection efficiency of the target DNA is high, and the expression level of the target protein is also high. The mass ratio of the target DNA to the transfection reagent is, for example, 1: 3.
in preferred embodiments, the transfection reagent comprises PEI (polyethyleneimine) or liposomes.
In a preferred embodiment, the transfection reagent is PEI. The PEI transfection reagent is a novel nano polymer transfection reagent. The transfection method can transfect various cell lines with high efficiency, including adherent cells and suspension cells, has higher transfection efficiency, and also has the characteristics of low toxicity, high efficiency, simple operation and the like. Furthermore, the cost of transient transfection of Expi293 cells is greatly reduced compared to liposomes for PEI.
In a preferred embodiment, the cells transiently transfected with Expi293 cells are cultured in Dynamis medium. The inventor firstly uses a CHO cell special culture medium Dynamis culture medium for culturing cells transiently transfected by Expi293 cells, the method not only ensures that the expression level of target protein cultured by using the Dynamis culture medium is equivalent to or even better than that of the existing Expi293 cell expression culture medium, but also ensures that the cost of the existing Expi293 cell culture medium is expensive, and the method greatly reduces the production cost of the target protein, thereby being easier to realize industrialization, being suitable for industrial production and being more beneficial to the fact that the Expi293 cells transiently transfect and express the target protein into daily operation.
The use of the above method for recombinant protein expression.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Part of the material information used in the embodiment of the present invention is as follows:
the plasmid R0202 is obtained by inserting a target gene (hPD-L1-ECD-mIgG1-FC) on the basis of a vector pCDNA3.4(thermo company); the plasmid R0208 is obtained by inserting a target gene (hPD-L2-ECD-mIgG1-FC) on the basis of a vector pCDNA3.4(thermo company); expi293 cells (ThermoFisher Scientific; A14635).
Expi293FTMExpression System Kit (gibco; A14635); dynamis medium (gibco; A2617502); PEI (available from Polysciences); liposomes (Gibco; Expifactamine 293 Transfection Kit).
Examples 1 to 6
Transfection assayPEI is adopted as an agent, plasmid R0208 is adopted as target DNA, and Dynamis culture medium is adopted for cells after transfection. The specific material dosage is as follows: the amount of target DNA was 75. mu.g, the amount of lipids was 250. mu.g, and the density of Expi293 cells was 2.9X 106cells/ml, transfection volume 50ml, culture volume 50 ml. The transfection method is as follows:
expi293 cells (ThermoFisher Scientific; A14635) were passaged one day before transient transfection using fresh Dynamis medium at 2X 106cells/ml were inoculated into 1L flasks (conning; 431147) at a density of cells/ml and placed in a cell culture shaker (Adolf Kuhner; ISF4-XC) at 37 ℃ with 8% CO2Culturing at 120 rpm.
On the day of transfection, Expi293 cells were counted with a cell counter (Countstar; IC1000), and diluted with fresh Dynamis medium to adjust the cell density to 2.9X 106cells/ml。
Preparing for transfection; mixing PEI and DNA for 5min, mixing the two gently for 20 times, and standing for 15-30min, not more than 30 min. Adding the DNA-PEI mixture to Expi293 cells, mixing, placing in a cell culture shaker (Adolf Kuhner; ISF4-XC) at 37 deg.C and 8% CO2Culturing at 120 rpm; the double antibody (gibco; 15140122) and the anticoagulant (gibco; 0010057) were added 4h after transfection.
The culture conditions of the transfected cells in examples 1-6 are shown in Table 1 below.
Comparative examples 1 to 4
Expi293 cells transfected with the target DNA used in comparative examples 1-4 were identical to those used in examples 1-6, except that the culture conditions of the transfected cells were as shown in Table 1 below:
TABLE 1
Test example 1
The supernatants from examples 1-8 and comparative examples 1-4 were harvested after the end of the transfected cell culture and tested for protein concentration by HPLC and protein activity by ELISA, as shown in Table 2 below:
TABLE 2
From the above table data, it can be seen that the temperature reduction at 33 ℃ is better than the temperature reduction at 31 ℃ in the treatment culture, and the cell growth rate, peak density and growth maintenance period are obviously better. The protein activities and expression amounts of the embodiments 1, 2 and 5 are all better, wherein the effect of the embodiment 5 is better, 76.85mg/L can be achieved, the protein activity is obviously improved, the defects of low instantaneous transfection efficiency and low protein expression amount and activity of Expi293 cells in the prior art are overcome, meanwhile, the technical scheme of the invention greatly reduces the production cost of target protein, enlarges the application range of the method, and is suitable for industrial production and daily operation.
Example 7
The transfection reagent used liposomes, target DNA used plasmid R0202, transfection according to Gibco kit instructions scheme, wherein, after transfection of cells using Dynamis medium for continuous culture for 7 days. The specific material dosage is as follows: the amount of target DNA was 10. mu.g, the amount of lipid was 27. mu.l, and the amount of Expi293 cells was 2.5X 107The transfection volume was 10ml and the culture volume was 10 ml.
Comparative example 5
The transfection reagent used was liposome and the target DNA used plasmid R0202, and transfection was performed according to the protocol of Gibco kit instructions, in which transfected cells were cultured for 7 days in Expi293 Expression medium (Gibco; A14635). The specific material dosage is as follows: the amount of target DNA was 10. mu.g, the amount of lipid was 27. mu.l, and the amount of Expi293 cells was 2.5X 107The transfection volume was 10ml and the culture volume was 10 ml.
Test example 2
After the transfected cells of example 7 and comparative example 5 were cultured, the supernatant was collected and subjected to HPLC to detect the protein concentration, and the results are shown in the following Table 3:
TABLE 3
| Peak area | Protein expression level (μ g/ml) | |
| Example 7 | 327 | 49.829 |
| Comparative example 5 | 293 | 44.648 |
As can be seen from the results of the above table, Dynamis and Expi293FTMExpression level of Expression media was comparable, but the price of Dynamis media was Expi293FTMAbout 1/5 of Expression media, the technical scheme of the invention greatly reduces the cost of expressing the target protein by transient transfection of the Expi293 cell.
Examples 8 to 19
The specific raw material information in examples 8-19 is shown in Table 4 below.
In examples 8-19, PEI was used as a transfection reagent, plasmid R0202 as a target DNA, and Dynamis medium was used as a cell culture medium, and the transfection method was as follows:
expi293 cells (ThermoFisher Scientific; A14635) were passaged one day before transient transfection using fresh Dynamis medium at 2X 106The cells were inoculated into 1L shake flasks (conning; 431147) at 37 ℃ in a cell culture shaker (Adolf Kuhner; ISF4-XC) with 8% CO2Culturing at 120 rpm.
On the day of transfection, Expi293 cells were counted with a cell counter (Countstar; IC1000), and diluted with fresh Dynamis medium to adjust the cell density to 2.9X 106cells/ml。
Preparing for transfection; PEI: DNA-4: 1-2: 1; mixing for 5min, mixing the two solutions gently for 20 times, and standing for 15-30min, not more than 30 min. Adding the DNA-PEI mixture to Expi293 cells, mixing, placing in a cell culture shaker (Adolf Kuhner; ISF4-XC) at 37 deg.C and 8% CO2Culturing at 120 rpm; the double antibody (gibco; 15140122) and the anticoagulant (gibco; 0010057) were added 4h after transfection.
The culture was continued for 7 days after transfection.
TABLE 4
| Plasmid R0202 | PEI | Expi293 cell | Transfection volume | Culture volume | |
| Example 8 | 10μg | 20μg | 2.9×107An | 10ml | 11ml |
| Examples9 | 10μg | 30μg | 2.9×107An | 10ml | 11ml |
| Example 10 | 10μg | 40μg | 2.9×107An | 10ml | 11ml |
| Example 11 | 15μg | 30μg | 2.9×107An | 10ml | 11ml |
| Example 12 | 15μg | 45μg | 2.9×107An | 10ml | 11ml |
| Example 13 | 15μg | 60μg | 2.9×107An | 10ml | 11ml |
| Example 14 | 20μg | 40μg | 2.9×107An | 10ml | 11ml |
| Example 15 | 20μg | 60μg | 2.9×107An | 10ml | 11ml |
| Example 16 | 20μg | 80μg | 2.9×107An | 10ml | 11ml |
| Example 17 | 25μg | 50μg | 2.9×107An | 10ml | 11ml |
| Example 18 | 25μg | 75μg | 2.9×107An | 10ml | 11ml |
| Example 19 | 25μg | 100μg | 2.9×107An | 10ml | 11ml |
Test example 3
Examples 8-19 supernatants were harvested after the end of the transfected cell cultures and subjected to HPLC for protein concentration, as shown in Table 5 below:
TABLE 5
From the above results, it can be seen that the amount of DNA is a major factor affecting the expression level of protein in a certain cell amount, and the expression level is increased by more than 50% compared with the conventional scheme (example 12) by optimizing the amount and ratio of DNA to PEI (examples 18-19). In addition, the cost of the liposome is more than 1000 times higher than that of PEI, when a certain amount of target protein is produced, the cost of the target protein transiently transfected and expressed by Expi293 cells is greatly reduced by using PEI as a transfection reagent compared with the liposome, and meanwhile, due to the adoption of a Dynamis culture medium in the culture medium, the method disclosed by the invention is easier to be daily operation, and the possibility of industrialization is provided.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (10)
1. A method for transiently expressing a target protein in Expi293 cells is characterized in that the Expi293 cells after transfection of target DNA are cultured, and the culturing step comprises cooling treatment.
2. The method of claim 1, wherein the step of culturing the Expi293 cells after transfection with the target DNA comprises: culturing at 36-38 deg.C, cooling to 32-34 deg.C for 1-3 days, and culturing for 4-10 days.
3. The method of claim 1, wherein the culturing step further comprises a replacement media resuspension process;
preferably, the step of culturing the Expi293 cells after transfection with the target DNA comprises: culturing at 36-38 deg.C, cooling to 30-34 deg.C for 1-3 days, culturing, changing culture medium for 3-5 days, and performing heavy suspension culture for 6-10 days;
preferably, the step of culturing the Expi293 cells after transfection with the target DNA comprises: culturing at 37 deg.C, cooling to 33 deg.C for culturing on day 2, changing culture medium on day 4, and performing resuspension culture until day 6.
4. The method according to claim 1, wherein the target DNA is transfected into Expi293 cells by a transfection reagent to obtain the Expi293 cells after transfection of the target DNA;
preferably, the ratio of the target DNA to the Expi293 cells is (15-30) μ g: (2X 10)7-5×107) And (4) respectively.
5. The method of claim 4, wherein the mass ratio of the target DNA to the transfection reagent is 1: (2-4).
6. The method of claim 4, wherein the mass ratio of the target DNA to the transfection reagent is 1: 3.
7. the method of claim 4, wherein the transfection reagent comprises PEI or liposomes.
8. The method of claim 7, wherein the transfection reagent is PEI.
9. The method of any one of claims 1-8, wherein the medium comprises Dynamis medium.
10. Use of the method of any one of claims 1-9 for recombinant protein expression.
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| CN114410682A (en) * | 2022-02-08 | 2022-04-29 | 美康生物科技股份有限公司 | Method for transient expression of target protein by 293T cell and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0309237A1 (en) * | 1987-09-25 | 1989-03-29 | Genentech, Inc. | A transient expression system for producing recombinant protein |
| US20050170450A1 (en) * | 2001-05-07 | 2005-08-04 | Yves Durocher | Enhanced production of recombinant proteins by transient transfection of suspension-growing mammalian cells |
| CN1820080A (en) * | 2002-12-23 | 2006-08-16 | 布里斯托尔-迈尔斯斯奎布公司 | Product quality enhancement in mammalian cell culture processes for protein production |
| CN101048511A (en) * | 2004-08-27 | 2007-10-03 | 惠氏研究爱尔兰有限公司 | Production of polypeptides |
| US20080145893A1 (en) * | 2006-09-17 | 2008-06-19 | Excellegene Sa | Method for producing a recombinant protein at high specific productivity, high batch yield and high volumetric yield by means of transient transfection |
| US20110039339A1 (en) * | 2008-05-15 | 2011-02-17 | Yves Durocher | Process, Vectors and Engineered Cell Lines for Enhanced Large-Scale Transfection |
| CN102533722A (en) * | 2010-12-24 | 2012-07-04 | 北京义翘神州生物技术有限公司 | Method for improving transient expression of recombinant proteins in mammalian cells through temperature jump |
| US20160222356A1 (en) * | 2013-08-30 | 2016-08-04 | Amgen Inc. | High Titer Recombinant AAV Vector Production in Adherent and Suspension Cells |
-
2019
- 2019-12-09 CN CN201911254039.5A patent/CN113025648B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5024939A (en) * | 1987-07-09 | 1991-06-18 | Genentech, Inc. | Transient expression system for producing recombinant protein |
| EP0309237A1 (en) * | 1987-09-25 | 1989-03-29 | Genentech, Inc. | A transient expression system for producing recombinant protein |
| US20050170450A1 (en) * | 2001-05-07 | 2005-08-04 | Yves Durocher | Enhanced production of recombinant proteins by transient transfection of suspension-growing mammalian cells |
| CN1820080A (en) * | 2002-12-23 | 2006-08-16 | 布里斯托尔-迈尔斯斯奎布公司 | Product quality enhancement in mammalian cell culture processes for protein production |
| CN101048511A (en) * | 2004-08-27 | 2007-10-03 | 惠氏研究爱尔兰有限公司 | Production of polypeptides |
| US20080145893A1 (en) * | 2006-09-17 | 2008-06-19 | Excellegene Sa | Method for producing a recombinant protein at high specific productivity, high batch yield and high volumetric yield by means of transient transfection |
| US20110039339A1 (en) * | 2008-05-15 | 2011-02-17 | Yves Durocher | Process, Vectors and Engineered Cell Lines for Enhanced Large-Scale Transfection |
| CN102533722A (en) * | 2010-12-24 | 2012-07-04 | 北京义翘神州生物技术有限公司 | Method for improving transient expression of recombinant proteins in mammalian cells through temperature jump |
| US20160222356A1 (en) * | 2013-08-30 | 2016-08-04 | Amgen Inc. | High Titer Recombinant AAV Vector Production in Adherent and Suspension Cells |
Non-Patent Citations (9)
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410682A (en) * | 2022-02-08 | 2022-04-29 | 美康生物科技股份有限公司 | Method for transient expression of target protein by 293T cell and application thereof |
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