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Application of Tet-off expression regulation system in cascade amplification of promoter activity

A technology of expression regulation and cascade amplification, applied in the application, the introduction of foreign genetic material using a vector, and the cells modified by the introduction of foreign genetic material, etc. Problems such as limited gene expression ability

Pending Publication Date: 2021-07-06
ZHUHAI UNITED LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing mature technology of lentivirus infection to construct protein expression cell lines, most of the lentiviruses obtained through the packaging of adherent cultured HEK 293T cells infect HEK 293T cells or other adherent culture cells; however, using adherent cells to express proteins There are the following defects: (1) the cell density is low, the expression ability of exogenous genes is limited, and the protein yield is low; (2) the culture medium with serum is required for cultivation, and there are risks such as pathogen contamination; (3) the adherent culture method is not conducive to large-scale Scale cultivation and industrial production
However, the yield of cell lines obtained by this stable cell line construction method is low, only 50-250 mg / L, which is far from meeting the rapidly developing production capacity requirements of contemporary biopharmaceuticals

Method used

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  • Application of Tet-off expression regulation system in cascade amplification of promoter activity
  • Application of Tet-off expression regulation system in cascade amplification of promoter activity
  • Application of Tet-off expression regulation system in cascade amplification of promoter activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Construction of pLUT-off-PEx-EGFP lentiviral recombinant plasmid,

[0075] Using the pLEGFP-C1 (ClonTech, Cat#6084-1) plasmid as a template, the EcoRI-EGFP-XhoI gene fragment was obtained by PCR amplification experiment, and the PCR primers were SeqPF and SeqPR.

[0076] SeqPF: 5'-ACCGGAATTCTCAGATCCGCTAGCGCTAC-3';

[0077] SeqPR: 5'-TGAGCTCGAGATCAGAGTCCGGACTTGTACAGC-3'.

[0078] Carry out EcoRI and XhoI double enzyme digestion on EcoRI-EGFP-XhoI gene fragment and pLUT-off plasmid (sequence shown in SEQ ID NO.5), and obtain EcoRI-EGFP with restriction endonuclease cohesive end by gel recovery and purification method -XhoI gene fragment and pLUT-off linearization vector. The pLUT-off linearization vector and the EcoRI-EGFP-XhoI gene fragment were connected by T4 DNA ligase, and the connection system and connection conditions were as follows: pLUT-off linearization vector 20ng, EcoRI-EGFP-XhoI fragment 200ng, T4 buffer (T4 Buffer) 2 μL, T4 DNA ligase 1 μL, ddH...

Embodiment 2

[0080] Example 2 Second-generation packaging system lentivirus packaging and infection

[0081] Process such as figure 2 Shown:

[0082] 1. Plasmid extraction: Use a plasmid extraction kit with endotoxin elimination effect to extract a large number of lentiviral recombinant vectors pLUT-off-PEx-EGFP and packaging plasmids psPAX2 (Addgene), pMD2.G (Addgene), and at the same time extract lentiviral positive controls Plasmid pLVX-IRES-ZsGreen1 (Clontech) is used to obtain high-concentration, high-purity, endotoxin-free plasmids required for virus packaging in the second-generation lentiviral packaging system.

[0083] 2. Cell culture: Resuscitate HEK 293T (ATCC), passage 3 times by conventional subculture method, prepare for co-transfection virus packaging experiment.

[0084] 3. Use the second-generation packaging system for lentivirus packaging:

[0085] (1) 24 hours before co-transfection, HEK 293T cells were treated with 0.3×10 5Cells / mL, 10mL DMEM complete medium (4mM L...

Embodiment 3

[0105] Example 3 Lentiviral Packaging and Infection of the Third Generation Packaging System

[0106] 1. Plasmid extraction: Use a plasmid extraction kit with endotoxin elimination effect to extract a large number of lentiviral recombinant vectors pLUT-off-PEx-EGFP and packaging plasmids pMDLg-PRRE (Addgene), pRSV-Rev psPAX2 (Addgene), pMD2.G (Adgene), while extracting the lentivirus positive control plasmid pLVX-IRES-ZsGreen1, to obtain the high-concentration, high-purity and endotoxin-free plasmid required for virus packaging in the third-generation lentivirus packaging system.

[0107] 2. Cell culture: resuscitate HEK 293T cells, and prepare for co-transfection virus packaging experiments after three times of subculture by conventional subculture methods.

[0108] 3. Use the third-generation packaging system for lentivirus packaging:

[0109] (1) 24 hours before co-transfection, HEK 293T cells were treated with 0.3×10 6 Cells / mL, 10mL DMEM complete medium was inoculated i...

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Abstract

The invention discloses an application of a Tet-off expression regulation system in cascade amplification of promoter activity. The Tet-off expression regulation system is the invention provided based on the condition that the inventor finds that the Tet-off expression regulation system has the effect of cascade amplification of the promoter activity, and the amplification effect on weak promoters is particularly obvious. By adopting the invention, a method for constructing an efficient expression stable cell strain by utilizing a lentivirus infection method is obtained. The method realizes short cell strain construction period, high efficiency, high expression and high stability, and provides a reliable basis for realizing large-scale and high-level expression of recombinant proteins.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the use of a Tet-off expression control system in cascade amplification promoter activity. Background technique [0002] Expressing recombinant proteins through stable mammalian cell lines is an important technology for the production of protein drugs in the field of biopharmaceuticals. However, the construction of stable cell lines using traditional transfection methods is a time-consuming and labor-intensive process. In traditional transfection technology, exogenous genes are randomly integrated in the host cell genome, resulting in obvious heterogeneity in gene copy number and integration site, so that the protein yield of the obtained engineered cell line is not high, and the long-term culture process can be difficult. Expression is unstable. [0003] Lentiviruses belong to the Retroviridae family and are named for the years-long incubation period of the viruses, th...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/67C12N5/10
CPCC12N15/86C12N15/67C12N2830/006C12N2740/15043
Inventor 韦苏珍李靖
Owner ZHUHAI UNITED LAB
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