Anti-human TNF-alpha stably-transformed cell strain as well as construction method and application thereof

A construction method and cell line technology, applied in the field of biomedicine, can solve the problems of low antibody production, chromosome loss, gene deletion, etc., and achieve stable antibody production, high antibody quality, and simple steps

Active Publication Date: 2020-04-03
ZHEJIANG ZHENGXI BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, fusion cell lines prepared by traditional methods are prone to chromosome loss and gene deletion, which seriously affect the stability between different batches in the production process of monoclonal antibodies and affect product quality.
In addition, fusion cell lines also have problems such as low antibody production and difficulty in cell culture

Method used

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  • Anti-human TNF-alpha stably-transformed cell strain as well as construction method and application thereof
  • Anti-human TNF-alpha stably-transformed cell strain as well as construction method and application thereof
  • Anti-human TNF-alpha stably-transformed cell strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] In this embodiment, a method for constructing an anti-human TNF-α stably transfected cell line, the method for constructing comprises the following steps:

[0036] (1) Prepare a plasmid containing the heavy chain gene of the anti-human TNF-α antibody and a plasmid containing the light chain gene of the anti-human TNF-α antibody, and co-transfect the parental CHO K1 cells with the above two plasmids;

[0037] Wherein, the nucleotide sequence of the anti-human TNF-α antibody heavy chain gene is shown in SEQ ID No.1, the nucleotide sequence of the anti-human TNF-α antibody light chain gene is shown in SEQ ID No.2, and the plasmid The preparation method was carried out according to the instructions of the QIAGEN kit; the transfection step of the plasmid was carried out according to the instructions of the Invitrogen kit.

[0038] (2) Perform MSX screening on the transfected CHO K1 cells to obtain stable transfected cells;

[0039] Specifically include:

[0040] (2.1) Cult...

Embodiment 2

[0054] In this example, the anti-human TNF-α stable transfection cell line CHO K1 obtained in Example 1 is used to produce anti-human TNF-α antibody. The production method includes the following steps:

[0055] (1) (a) Transfer the anti-human TNF-α stably transfected cell line CHO K1 (hereinafter referred to as cell line CHO K1) to a 125mL shake flask containing 30mL of complete CD culture medium, and place the shake flask at 37°C, 5% CO 2 In the shaker, shake culture at 120rpm;

[0056] The cryopreserved anti-human TNF-α stably transfected cell line CHO K1 needs to be resuscitated in advance. The recovery method is as follows: take the cell line CHO K1 out of the liquid nitrogen tank and thaw it quickly in a 37°C water bath, then centrifuge the cell solution, Remove the supernatant, then wash with PBS;

[0057] During the initial culture, subculture the cell line CHO K1 every 2-3 days, at least 2 times, and pay attention to avoid excessive cell density during the subculture...

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Abstract

The invention discloses an anti-human TNF-alpha stably-transformed cell strain as well as a construction method and application thereof. The stably-transformed cell strain is called as an anti-human TNF-alpha stably-transformed cell strain CHO K1, preserved in China Center For Type Culture Collection (CCTCC) on 25th, September, 2019; and a preservation address of the stably-transformed cell strainis Wuhan university, Wuhan City, China, and a preservation number is CCTCC No:C2019183. The yield of the anti-human TNF-alpha antibody of the anti-human TNF-alpha stably-transformed cell strain CHO K1 can be up to 3g/L, and is 6 times of existing anti-human TNF-alpha antibody expression cell strains; and the antibody yield in different production batches is stable, and the antibody quality is high.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to an anti-human TNF-α stably transfected cell line and its construction method and application. Background technique [0002] TNF (Tumor necrosis factor, tumor necrosis factor) is a cytokine that occurs naturally in inflammation and immune response. Studies have found that in the synovial fluid of patients with rheumatoid arthritis, the level of TNF is increased, and it is also involved in pathological inflammation. and joint damage play an important role. [0003] Adalimumab can specifically bind to TNF-α, but not to cytokines such as TNF-β, and block the interaction between TNF-α and TNF receptors on the surface of p55 and p75 cells, reducing the overproduction of TNF-α The inflammatory response triggered by the release of TNFα has important clinical significance for the treatment of diseases caused by the excessive production of TNFα. Adalimumab is a self-inject...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/13C12N15/85C07K16/24C12R1/91
CPCC07K16/241C12N15/85
Inventor 张洋
Owner ZHEJIANG ZHENGXI BIOMEDICAL CO LTD
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