Transient Transfection Process of Drosophila Cells

A transient transfection, Drosophila technology, applied in the field of bioengineering, can solve the problems of limiting the application of TGE technology, and the protein expression yield of insect cells cannot achieve the expected results.

Active Publication Date: 2021-03-23
FOSHAN CANTON BIOLOGICS CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Insect cell-based TGE technology has also been introduced (Loomis et al., 2005; Shen et al., 2013), but at present, due to the lack of efficient TGE operating procedures with process optimization, the yield of protein expression in insect cells based on TGE technology cannot be achieved. Expected effect, limiting the application of TGE technology in insect cells

Method used

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  • Transient Transfection Process of Drosophila Cells
  • Transient Transfection Process of Drosophila Cells
  • Transient Transfection Process of Drosophila Cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 provides a method for expressing the secreted protein TNFR-Fc using the technique of transient transfection of Drosophila cells of the present invention. The parameters used in this example have been optimized. It can be understood that in other examples, Not limited to this.

[0044] Dilute Drosophila S2 cells at 1×10 6 The density of Drosophila cells / mL was subcultured into Sf900II SFM medium, and shake culture was carried out at 28°C and 280rpm → cultured for 2 days to logarithmic production phase, centrifuged and resuspended to a density of 15×10 6 Drosophila cells / mL→according to 0.6μg / 10 6 pDNA of a Drosophila cell and 2.0 μg / 10 6 PEI of Drosophila cells was added to the transfection system, and the cells were diluted to 5×10 after 1 hour. 6 Drosophila cells / mL → after 5 days of culture, the protein expression level was detected by ELISA.

[0045] The process can be scaled up from 10mL to 300mL and beyond. The specific process of expressing TNFR-Fc ...

Embodiment 2

[0059] Example 1 provides a method for expressing the intracellular protein EGFP using the technique of transient transfection of Drosophila cells. The parameters used in this example have been optimized, and it is understandable that in other examples, it is not limited thereto.

[0060] Dilute Drosophila S2 cells at 1×10 6 The density of Drosophila cells / mL was subcultured into Sf900II SFM medium, and shake culture was carried out at 28°C and 280rpm → cultured for 3 days to logarithmic production phase, centrifuged and resuspended to a density of 15×10 6 Drosophila cells / mL→according to 0.6μg / 10 6 pDNA of a Drosophila cell and 2.0 μg / 10 6 PEI of Drosophila cells was added to the transfection system, and the cells were diluted to 5×10 after 1 hour. 6 Drosophila cells / mL → after 3 days of culture, detect the percentage of EGFP-positive cells by GUAVAEasyCyte flow cytometer, and perform fluorescence quantification by TECAN SaphirelI fluorescence photometer.

[0061] The proc...

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Abstract

The invention relates to a transient transfection process of drosophila melanogaster cells. The transient transfection process comprises the following steps: adding a transfection reagent and plasmids containing target DNA to a drosophila melanogaster cell suspension, and carrying out transient transfection, wherein the transfection reagent is cationic polymer PEI, the adding amount of the transfection reagent is 0.8-3.2 [mu] g / 10<6> drosophila melanogaster cells, and the adding amount of the plasmids containing target DNA is 0.2-1.4 [mu] g / 10<6> drosophila melanogaster cells; and carrying out culturing to obtain the drosophila melanogaster cells transfected with the plasmids containing the target DNA. The transient transfection process of the drosophila melanogaster cells is easy to operate, the used transfection reagent is low in cost, and the transfection is rapid. For the transient transfection process of the drosophila melanogaster cells, through the transient transfection technology easy to operate, the low-cost and high-yield target DNA expression can be realized in the drosophila melanogaster cells within a short time, meanwhile, the process can be applied to the large-scale industrial production, and thus the technical difficulty that the insect cells can not easily realize the industrialization production of recombinant protein through the transient transfection process is overcome.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a technique for transient transfection of fruit fly cells. Background technique [0002] The insect cell expression system has been widely used for the expression of many human recombinant proteins including tissue factor and antibodies (Kosr et al., 1997). Drosophila Schneider 2 (Drosophila Schneider 2) cells are derived from the late stage of Drosophila embryos and began to be used commercially in the 1990s, mainly through the transfection of specific plasmids to select transfected cell pools and further obtain cell lines for protein expression. This technology is an important alternative technology of the baculovirus expression vector system (BEVS), and has been successfully applied to the expression of active receptor proteins, ion channels, cytoskeleton proteins, transcriptional regulators, hormones and coagulation factors (Millar et al. Millar et al., 1994; Tota et al., 1995;...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85
CPCC12N15/85C12N2800/105
Inventor 沈潇林兴华林泽斌景涛
Owner FOSHAN CANTON BIOLOGICS CO LTD
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