Gene sequence capable of preventing gradual inactivation of promoter in subculture and applications of gene sequence

A technology of gene sequence and culture medium, which is applied in the field of gene sequence to prevent the gradual inactivation of promoters during subculture, can solve the problems affecting the yield of monoclonal antibodies, and can not achieve high yield and low cost of monoclonal antibodies, so as to reduce production The effect of high cost and high output

Inactive Publication Date: 2016-01-20
BEIJING YISHENGHE BIOLOGICAL SCI & TECHCO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] In the subculture of general CHO cells, the promoter will be gradually inactivated, which will affect t

Method used

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  • Gene sequence capable of preventing gradual inactivation of promoter in subculture and applications of gene sequence
  • Gene sequence capable of preventing gradual inactivation of promoter in subculture and applications of gene sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The genomic DNA of CHO cells was incompletely digested with EcoRI endonuclease, and the resulting fragments were randomly connected to the expression vector for screening:

[0049] The screening method is:

[0050] 1) Put 0.5×10 5 ~2×10 5 (A) CHO cells were seeded on a culture plate and added to complete medium without antibiotics;

[0051] 2) Prepare the complex: Dilute each DNA and Lipofectamine2000 obtained by incomplete digestion of CHO cells in a serum-free and antibiotic-free culture medium, mix them, incubate at room temperature for 5 minutes, mix the two, mix well, and incubate at room temperature 20 minutes;

[0052] 3) Aspirate the culture medium from the culture plate and wash the cells with serum-free medium;

[0053] 4) Add the compound evenly to the culture plate;

[0054] 5) Put the culture plate into the incubator and incubate for 4-6 hours;

[0055] 6) Observe the expression of the transferred gene after 24 to 48 hours: take the culture supernatant and detect the ...

Embodiment 2

[0058] Insert the aforementioned sequence into the vector (pCOCKCMVwt, synthesized by the company itself, the sequence is from addgene): 0.5~2×10 1 day before transfection 5 Adalimumab-producing CHO cells were seeded on a 24-well culture plate, and 500 μl of complete medium without antibiotics was added to ensure that the cell confluence reached 90-95% during transfection. Dilute 0.8μg plasmid DNA (pCOCKCMVwt, synthesized by our company, sequence derived from addgene) in 50μl serum-free and antibiotic-free culture medium and mix gently; dilute 2μl Lipofectamine2000 in 50μl serum- and antibiotic-free culture medium and mix gently Evenly, incubate at room temperature for 5 minutes. Note: It must be done within 25 minutes; after 5 minutes, mix them and mix gently, and incubate at room temperature for 20 minutes. Aspirate the culture medium from the culture plate, and wash the cells twice with serum-free medium. Add the complex (100 μl total volume) to the culture wells, shake the...

Embodiment 3

[0063] Insert the aforementioned sequence into the vector (pCOCKCMVwt, synthesized by the company itself, the sequence is from addgene): 0.5~2×10 1 day before transfection 5 Adalimumab-producing CHO cells were seeded on a 24-well culture plate, and 500 μl of complete medium without antibiotics was added to ensure that the cell confluence reached 90-95% during transfection. Dilute 0.8μg plasmid DNA (pCOCKCMVwt, synthesized by our company, sequence derived from addgene) in 50μl serum-free and antibiotic-free culture medium and mix gently; dilute 2μl Lipofectamine2000 in 50μl serum- and antibiotic-free culture medium and mix gently Evenly, incubate at room temperature for 5 minutes. Note: It must be done within 25 minutes; after 5 minutes, mix them and mix gently, and incubate at room temperature for 20 minutes. Aspirate the culture medium from the culture plate, and wash the cells twice with serum-free medium. Add the complex (100 μl total volume) to the culture wells, shake the...

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Abstract

The invention relates to molecular biology and particularly discloses a gene sequence capable of preventing gradual inactivation of a promoter in subculture and applications of the gene sequence. The nucleotide sequence of the gene sequence is shown as SEQ ID No.1. The gene sequence is used for constructing a carrier and a CHO cell line, can prevent gradual inactivation of the promoter in subculture of CHO cells and can be used for monoclonal antibody production through the CHO cell line to increase the monoclonal antibody yield.

Description

Technical field [0001] The invention relates to molecular biology, in particular to a gene sequence that prevents the promoter from being gradually inactivated in subculture and its application. Background technique [0002] Biopharmaceuticals is the fastest growing industry in the pharmaceutical industry in recent years. As the most eye-catching product category in biopharmaceuticals, monoclonal antibodies have experienced a growth rate of 41% in the past 15 years. The indications of monoclonal antibodies are mainly focused on tumors and autoimmune diseases, in addition to anti-infection, ophthalmology, osteoporosis, myocardial infarction, blood system diseases and so on. At the same time, it is more and more favored by patients because of its strong targeting and low side effects. In 2013, 6 of these monoclonal antibody drugs were ranked in the top 8 of the global prescription drug sales rankings. [0003] Compared with the first and second-generation biotechnology medical prod...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85C12N5/10C07K16/00
Inventor 狄春辉刘进王晨膺孙利燕吕红霞范林霞
Owner BEIJING YISHENGHE BIOLOGICAL SCI & TECHCO LTD
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