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Method for improving transient expression of recombinant proteins in mammalian cells through temperature jump

A temperature mutation, mammalian technology, applied in the biological field, can solve the problem of not enhancing the transient transfection efficiency of cells, achieve great commercial value, and improve the effect of transfection efficiency

Active Publication Date: 2012-07-04
北京义翘神州科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many studies on the application of temperature regulation and protein expression, there is no report on the use of transient temperature mutations in the transient transfection process to enhance the transient transfection efficiency of cells

Method used

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  • Method for improving transient expression of recombinant proteins in mammalian cells through temperature jump
  • Method for improving transient expression of recombinant proteins in mammalian cells through temperature jump
  • Method for improving transient expression of recombinant proteins in mammalian cells through temperature jump

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: The influence of temperature mutation on HEK 293 cell recombinant EGFR protein expression

[0052] In order to analyze the impact of the mutation of the temperature of the cell environment during transfection on the transient transfection process of the cells, we used the plasmid encoding the EGFR protein with the Fc tag to perform the following experiments. One day before transfection, a total of 9 flasks of HEK 293 cells were inoculated and cultured in a shaker at 36.5°C. Three hours before transfection, 3 bottles were taken respectively and placed in a shaker at a temperature of 31°C and 39°C, and the other 3 bottles were continued to be placed in a shaker at 36.5°C. Transfection was performed 3 hours later. After adding the exogenous DNA-transfection reagent complex, the shake flasks were put back into the shakers at 31°C, 36.5°C and 39°C respectively. After 3 hours, all shake flasks were placed in a shaker at 36.5°C for incubation. All cell DNA-tra...

Embodiment 2

[0054] Example 2: Determination of the start time of temperature mutation in HEK 293 cells

[0055] The following experiment was designed to determine the start time of temperature mutation of HEK 293 cells: one day before transfection, a total of 18 flasks of HEK 293 cells were inoculated, divided into 6 groups, with 3 parallel samples in each group, and cultured in a shaker at 36.5°C. On the next day, 4 hours, 3 hours, 2 hours, and 1 hour before transfection, a group of three shake flasks were taken out from the shaker at 36.5°C and placed in a shaker at 39°C. These 4 groups of cells continued to be placed in a shaker at 39°C for 3 hours after the DNA-transfection reagent complex was added to the cell culture medium, and then transferred to a shaker at 36.5°C to continue culturing. The cells in group 5 (ie, 0 hour group) were placed in a shaker at 36.5°C before transfection, and the DNA-transfection reagent complex was added to the cell culture medium and placed in a shaker ...

Embodiment 3

[0057] Example 3: Determination of duration of temperature mutation in HEK 293 cells

[0058]The following experiment was designed to determine the duration of the temperature mutation of HEK 293 cells: one day before transfection, a total of 18 flasks of normally treated cells were divided into 6 groups, with 3 parallel samples in each group, and they were cultured normally in a shaker at 36.5°C. Two hours before the staining, the five groups of cells were taken out from the shaker at 36.5°C and placed in a shaker at 39°C for culture. After 2 hours of transfection, after the DNA-transfection reagent complex was added to the cell culture medium, one group was taken and returned to a shaker at 36.5°C to continue culturing (ie, the 0-hour group). The other 4 groups were cultured in the shaker at 39°C, and one group was taken back to the shaker at 36.5°C at 1 hour, 2 hours, 3 hours and 4 hours after the DNA-transfection reagent complex was added to the cell culture medium. Conti...

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Abstract

Provided is a method for improving transient expression of recombinant proteins through improvement of transfection efficiency, wherein the transfection efficiency is improved through changing of environment of cells before and after transfection. The method enables expression index of the transient transfection recombinant proteins to be improved by 37% to 56%.

Description

technical field [0001] The invention relates to the field of biotechnology, and specifically provides a new method for improving the transient transfection expression of recombinant proteins in mammalian cells. Background technique [0002] Systems for the production of recombinant proteins using mammalian cells have been established for a long time. Compared with the lower eukaryotic cell expression system or prokaryotic cell expression system, the recombinant protein produced by the mammalian cell system has the ability to optimally express high molecular weight protein products, make complex structural proteins produce correct folding and give glycoproteins Provide suitable post-translational modification functions and become the best choice for expressing recombinant proteins in the field of biopharmaceuticals [Chen P, Hutter D, Liu P, et al. Protein Expr Purif, 2002; 24(3); 481-488]. [0003] The traditional mammalian cell expression system is to construct a stable cel...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N5/10
Inventor 马宁宁张延静潘范彬王文志谢良志
Owner 北京义翘神州科技股份有限公司
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