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Construction method for single copy cell strain of stably and highly expressed protein and application of single copy cell strain

A construction method and cell line technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc.

Active Publication Date: 2021-01-05
HYQUO MOLECULE BEIJING TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is no technical solution and technical inspiration for constructing 293T cells with stable and high-efficiency expression and replaceable target gene by random screening strategy

Method used

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  • Construction method for single copy cell strain of stably and highly expressed protein and application of single copy cell strain
  • Construction method for single copy cell strain of stably and highly expressed protein and application of single copy cell strain
  • Construction method for single copy cell strain of stably and highly expressed protein and application of single copy cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of a high protein expression system based on 293T cells and its optimization

[0029] The transient expression plasmid expressing anti-TNFα single chain antibody is mainly used in the experiment

[0030] pCDNA3.1-hygro-SV40-DHFR-SV40 polyA-FRT-TNF-Loxp (abbreviated as pCD), double histone expression plasmid (pF2AC) and pFRT-EGFP-LoxP (pCDFGL) and pFRT- RFP-LoxP (pFRL) replacement plasmid (Chen C, Li N, Zhao Y, et al.Coupling recombinase-mediated cassette exchange with somatic hypermutation for antibody affinity maturation in CHOcells[J].Biotechnology&Bioengineering,2016,113(1):39- 51.).

[0031] The cell line used in the experiment is 293T cells (human embryonic kidney epithelial cell line) and various subcellular clones constructed on the basis of it: use 10% calf serum (Hyclone) and 100U / mL double antibody (penicillin and streptomycin) in DMEM medium (Invitrogen, CA) at 37°C in 5% CO 2 Adherent culture in the incubator.

[0032] In this stud...

Embodiment 2

[0043] Embodiment 2 The first round of recombinant replacement

[0044] The pFRT-EGFP-LoxP (pCDFGL) replacement plasmid and the recombinase plasmid pF2AC (expressing Flp and Cre dual histase) were co-transfected into 293T-anti-TNF cells, and under the combined action of Flp and Cre enzymes, the GFP gene in the cells was replaced anti-TNFα antibody gene. Replacement of successful cells transcriptionally expresses GFP protein, thereby autofluorescing green, as in figure 2 a. The results of the first round of recombinant replacement by flow cytometry showed that: the ordinate represents the display level of anti-TNFα antibody, and the abscissa represents the replacement level of GFP protein. After replacement, the GFP-positive cells were divided into two groups, one group only contained GFP fluorescence, and the other group contained double fluorescence signals of GFP and labeled anti-TNFα antibody (indicating that some cells contained multiple replacement sites, and some site...

Embodiment 3

[0046] Embodiment 3 The second round of recombinant replacement

[0047] As in Example 2, the pFRT-RFP-LoxP (pFRL) replacement plasmid and the recombinase plasmid pF2AC were co-transfected into 293T-GFP cells, and the RFP gene replaced GFP in the cells under the joint action of Flp and Cre enzymes. Replacement of successful cell transcription and expression of RFP protein makes the cell spontaneously emit red fluorescence, such as image 3 a. The results of the second round of recombinant replacement by flow cytometry showed that the abscissa represents the expression level of GFP protein, and the ordinate represents the replacement level of RFP. RFP-positive cells were divided into two groups, one group containing only RFP fluorescence, and the other group containing dual fluorescence of GFP and RFP, and flow cytometry enriched cells with only RFP fluorescence (such as image 3 Gate R of B), named 293T-RFP.

[0048] In order to obtain a dominant cell population with strong...

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Abstract

The invention provides a construction method for a single copy cell strain of stably and highly expressed protein and application of the single copy cell strain. The method is based on target genes, which can be rapidly and accurately inserted and can be stably expressed a at a high level, of a 293T cell. According to the method, a 293T cell strain which is stably and efficiently expressed and canreplace the target genes is established through random insertion combined with a high-throughput screening strategy. Based on the cell strain, adenosine A2A receptor protein (ADORA2A) belonging to one of GPCR families is successfully expressed. By utilizing such method, repeated cassette-type recombination replacement can be carried out, an antibody or other protein genes to be expressed or optimized can be rapidly and accurately inserted into a cell gene cassette, and the integrity of the gene cassette cannot be broken.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a single-copy cell line with stable and highly expressed proteins and its application. The method is based on 293T cells that can be inserted quickly and accurately and can stably express a target gene at a high level. Background technique [0002] Antibody drug research has been booming in recent years. Since 2010, the number of monoclonal antibodies entering late-stage clinical research has increased rapidly every year. One of the key technologies driving the development of therapeutic antibodies is affinity maturation of antibodies in vitro. Currently, technologies widely used in in vitro antibody affinity optimization include ribosome display, phage display, yeast display, bacterial display, and mammalian cell display technologies. Since the antibodies screened by mammalian cell display technology are closer to human proteins in post-translational modif...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12N5/10G01N15/14
CPCC12N15/85C12N15/65C12N5/0686C07K14/70571G01N15/14C12N2800/107C12N2510/02
Inventor 杭海英蒋薇赵云安莉莉
Owner HYQUO MOLECULE BEIJING TECH CO LTD
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