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CHO-DHFR <+> cell strain and application thereof

A technology of CHO-DHFR and cell lines, which is applied in the direction of animal cells, reproductive tract cells, interleukin, etc., can solve the problems of increasing the workload of screening cell clones, degradation products that are unfavorable for cell growth, and high cost of use

Active Publication Date: 2021-03-16
KANGLITAI BIOMEDICAL (QINGDAO) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this system also has some shortcomings, such as the method requires a higher concentration of MTX, and the expression level of foreign genes is not high, which increases the workload of screening cell clones, etc.
[0007] ② Glutamine synthetase (GS) amplification system (representing cells CHOK1SV-KO (Lonza), CHOZN (Merk), CHO-S (Thermo)) is a more effective system developed recently, with higher amplification efficiency , high speed, good stability, and high yield. At the same time, the system does not require the addition of additional glutamine, resulting in lower content of metabolic waste ammonia, better cell viability, and more conducive to subsequent industrial production. But the use cost is higher
This method knocks out the endogenous GS gene of CHO cells. Therefore, a large amount of glutamine needs to be added to the medium in the subsequent culture process. However, glutamine is unstable and can be decomposed by about 7 days at 4°C. 50%, and the degradation products are not conducive to cell growth

Method used

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  • CHO-DHFR &lt;+&gt; cell strain and application thereof
  • CHO-DHFR &lt;+&gt; cell strain and application thereof
  • CHO-DHFR &lt;+&gt; cell strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: CHO-DHFR+ cell line screening process

[0046] This embodiment provides a CHO-DHFR+ cell line, the CHO-DHFR+ cell line is obtained by screening by the following method:

[0047] (1) Resuscitate CHO-DG44 cells (obtained from the market) for subculture adaptation, and prepare for transfection when the cell growth status returns to normal.

[0048] (2) On the day of transfection, CHO-DG44 cells were washed by OptiMEM centrifugation and resuspended to 3.0×10 6 cells / ml, and then add the cells to a 6-well plate at 0.8ml / well for later use.

[0049] (3) Mix DNA (per well), add 2.5 μg of DNA (pSV2-DHFR plasmid) into 100 μL of OptiMEM medium, and mix gently. For the blank wells, 2.5 μL of PBS was used instead of DNA, and the mixture was incubated at room temperature for 30 min.

[0050] The information of the pSV2-DHFR plasmid is shown in Table 1 below:

[0051] Table 1:

[0052]

[0053]

[0054] The nucleotide sequence of the pSV2-DHFR plasmid is shown i...

Embodiment 2

[0069] Example 2: Comparative evaluation of the growth performance of CHO-DHFR+ cell lines

[0070] The CHO-DHFR+ cell line constructed and preserved in Example 1 of the present invention and the commercially available CHO-S cell line were subjected to a comparison experiment of cell growth performance evaluation, as follows:

[0071] (1) resuscitating the CHO-DHFR+ cell line of Example 1 of the present invention and the commercially available CHO-S cell line (control group) in fresh CD OptiCHO medium (containing 3mM glutamine) for subculture adaptation, to the cell The growth status returned to normal.

[0072] (2) Cells recovered to normal growth state were treated with 0.5×10 6 The cells / ml density was inoculated into 125ml shake flasks, 30ml per bottle.

[0073] (3) The day of inoculation is recorded as day0, and 50 μl of cell suspension is taken every day after inoculation to test the cell viability and density on a cell counter, and the test data of day1, day2, day3......

Embodiment 3

[0076] Example 3: Construction of engineered cell lines highly expressing recombinant IL-10

[0077] This embodiment provides a method for constructing an engineered cell line that highly expresses recombinant IL-10, which includes the following steps:

[0078] (1) Resuscitate the CHO-DHFR+ cell line of Example 1 of the present invention into fresh CD OptiCHO medium for subculture adaptation until the cell growth state returns to normal.

[0079] (2) Cells recovered to normal growth state were treated with 0.5×10 6 The cells / ml density was inoculated into 125ml shake flasks, 30ml per bottle.

[0080] (3) Link the optimized IL-10 gene (its nucleotide sequence as shown in SEQ ID NO: 1) to the pcDNA3.1-beta plasmid to construct and obtain the recombinant plasmid vector (its nucleotide sequence as shown in SEQ ID NO: 1) NO: 7).

[0081] The pcDNA3.1-β plasmid is as follows:

[0082] The replicon of the pcDNA3.1-β plasmid is pUC origin, the promoter is CMV promoter, the resista...

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Abstract

The invention provides a CHO-DHFR <+> cell strain and an application thereof. The preservation number of the cell strain is CCTCC (China Center For Type Culture Collection) NO: C2019264. The CHO-DHFR<+> cell strain can be suspension-cultured at high density without serum, normal growth and production of cells can be maintained only by adding a small amount of glutamine into a culture system in the culture process, accumulation of toxic waste NH4 <+> generated by glutamine metabolism is reduced, the cell doubling level is high, and the cell viability is high. A pcDNA3.1-beta recombinant plasmid vector containing an IL-10 gene sequence is transfected into the CHO-DHFR <+> cell strain to obtain an engineering cell strain for expressing recombinant IL-10, which is cultured in a CD OptiCHO medium containing G418, and efficiently express the recombinant IL-10. The transient expression quantity of the recombinant IL-10 protein is greater than 0.96 mg / L, so that the CHO-DHFR <+> cell strain has relatively good commercial application value.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a CHO-DHFR+ cell line and an application thereof. Background technique [0002] Chinese hamster ovary (CHO) cells are the preferred host cells for the production of protein drugs, because compared with other systems, CHO cells have the following advantages: ①CHO cells have accurate processing and modification functions for proteins, so the biological properties of the expressed proteins The activity is closer to the natural protein; ②CHO cells have relatively strong ability to withstand shear force and osmotic pressure, and can choose the method of adherent culture or suspension culture according to the culture requirements; ③The cells after the integration of foreign genes are stable, and the recombinant genes It can be amplified and expressed efficiently; ④The expressed target protein can be transported from the cell to the outside of the cell, and CHO cells only expre...

Claims

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Application Information

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IPC IPC(8): C12N5/10C07K14/54C12N15/85
CPCC12N5/0682C07K14/5428C12N15/85C12N9/003C12Y105/01003C12N2800/107C12N2510/02
Inventor 乔磊杨盼盼郑彬彬王建刚吴明远
Owner KANGLITAI BIOMEDICAL (QINGDAO) CO LTD
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