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Culture medium for cloning formation of Chinese hamster ovary cells

A medium and a technology for screening medium, applied in germ cells, culture process, tissue culture, etc., can solve the problem of being under the regulation of very late virus promoters

Pending Publication Date: 2021-07-16
SHANGHAI SINOMAB BIOTECHNOLOGY CO LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expressed protein can have correct folding, disulfide bond matching, post-translational processing modification, and can also accommodate macromolecular inserts, and can express multiple genes at the same time, but the disadvantage is that the expression of foreign proteins is at a very late stage under the control of a viral promoter

Method used

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  • Culture medium for cloning formation of Chinese hamster ovary cells
  • Culture medium for cloning formation of Chinese hamster ovary cells
  • Culture medium for cloning formation of Chinese hamster ovary cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, screening method

[0028] The Chinese hamster ovary cell CHO used in the present invention is derived from CHO-K1, and the clone screening of monoclonal antibody expression is carried out using this host cell. The cell culture temperature was 37°C, and the content of carbon dioxide was 5%.

[0029] Synthesize the antibody gene sequence to be expressed, prepare an expression vector containing the antibody gene sequence, transfect CHO-K1 host cells, carry out cell culture, and clone screening.

[0030] The screening of CHO cell clones is carried out by the limited dilution method, and the medium CHOM-C2-1, CHOM-C2-5, and CHOM-C2-6 without serum and animal-derived components are used for the cultivation of CHO cells, which are serum-free and animal-derived The cloning medium CHOM-C2-1, CHOM-C2-5, and CHOM-C2-6 are beneficial to the survival and growth of Chinese hamster ovary cell lines, and can replace the traditional culture method using 10% fetal bovine s...

Embodiment 2

[0047] Embodiment 2, cloning control

[0048] After culturing the CHO cell clones using different cloning media, the expression levels of proteins expressed by the obtained clones were determined. Use the dot blot method to determine the protein expression level. When the clone is cultured for 12 days, draw 1.5ul of the cell culture supernatant from the 96-well plate and put it on the nitrocellulose (NC) membrane. After standing for 5min, put it in 10% skimmed milk powder at room temperature Incubate for 2h. Wash twice with phosphate-buffered saline (PBS), 3-5 minutes each time; add 1:1000 diluted antibody, and incubate at room temperature for 1 hour. After washing 5 times with phosphate buffered saline (PBS) containing 0.1% Tween20, place it in DAB color developing solution for 3-5 minutes to develop color.

[0049] The protein expression results of clones obtained from different cloning media are as follows: image 3 shown. Use different cloning media to culture CHO cell...

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Abstract

The invention provides a culture medium for cloning formation of Chinese hamster ovary cells. The culture medium provided by the invention does not contain serum or animal-derived components. The cloning culture medium provided by the invention can enable the CHO cells to be more compact in cloning agglomeration and less in diffusion, the growth speed of cloning is fast, and expression clones suitable for production and use purposes can be more efficiently selected.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a culture medium for the formation of Chinese hamster ovary cell clones. Background technique [0002] For the production of existing biological products, different types of production expression systems for biological products are selected according to various factors such as production purpose and cost control. Commonly used expression systems for the production and expression of biological products include E. coli expression systems, yeast expression systems, insect expression systems, and mammalian cell expression systems. [0003] Escherichia coli expression system is characterized by clear genetic background, fast propagation, low cost, high expression level, easy purification of expressed products, good stability, strong anti-pollution ability, and wide application range. [0004] The yeast expression system is characterized by the fact that yeast is a ...

Claims

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Application Information

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IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2500/20C12N2500/22C12N2500/32C12N2500/38C12N2500/40C12N2500/46
Inventor 侯盛郭怀祖郭清城徐进戴建新聂丽张存超寇庚黄卫红
Owner SHANGHAI SINOMAB BIOTECHNOLOGY CO LTD
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