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Method for improving IgG1 antibody yield

A high-yield, antibody-based technology, applied in the biological field, to achieve the effect of increasing expression and increasing yield

Active Publication Date: 2020-01-10
天津大学前沿技术研究院
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at this stage, the yield is only optimized from the perspective of the fermentation process and the monoclonal antibody itself, and no one has studied the influence of the tricarboxylic acid cycle pathway of E. coli itself (as shown in Figure 1) on the monoclonal antibody yield

Method used

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  • Method for improving IgG1 antibody yield
  • Method for improving IgG1 antibody yield
  • Method for improving IgG1 antibody yield

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Embodiment 1

[0019] A method for improving IgG1 antibody production, comprising the steps of:

[0020] (1) In vitro synthesis and amplification of genes:

[0021] From the NCBI database, obtain the light chain of IgG1 derived from cetuximab (Cetuximab) and the heavy chain of IgG1, the amino acid sequences of the light chain and heavy chain of IgG1 are respectively on the JCAT website, by input: avoid XbaI, NdeI, The SpeI and HindIII restriction enzyme sites were used as conditions to obtain the nucleotide sequences of the optimized IgG1 light chain and the optimized IgG1 heavy chain, which were synthesized and amplified in vitro; and the optimized IgG1 light chain Linked with the optimized heavy chain to form a nucleotide sequence;

[0022] The amino acid sequence of the light chain of IgG1 is shown in SEQ ID NO.1;

[0023] The amino acid sequence of the heavy chain of IgG1 is shown in SEQ ID NO.3;

[0024] The nucleotide sequence of the light chain of the optimized IgG1 is shown in SEQ...

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Abstract

The invention discloses a method for improving IgG1 antibody yield, which comprises the following steps: (1) gene in-vitro synthesis and amplification: obtaining optimized nucleotide sequences of a light chain and a heavy chain of lgG1, and connecting the nucleotide sequences into a nucleotide sequence; (2) connecting the nucleotide sequence to a p2A4 vector to obtain p2A4-HL; (3) designing and assembling sRNA; (4) respectively connecting the sRNA sequence connected to a sRNA receiver to the p3C5 vector; (5) obtaining the recombinant bacterium 1, the recombinant bacterium 2, the recombinant bacterium 3, the recombinant bacterium 4 and the recombinant bacterium 5 by respectively performing introducing into escherichia coli together with p2A4-HL; and (6) respectively fermenting the recombinant bacteria to obtain the high-yield IgG1 antibody. According to the method disclosed by the invention, the expression quantity of the monoclonal antibody in escherichia coli can be increased, relatedenzymes of a tricarboxylic acid circulating metabolic pathway can be inhibited in a targeted manner, and more amino acids for synthesizing redundant enzymes can be used for synthesizing the IgG1 antibody, so that the yield of IgG1 is increased.

Description

technical field [0001] The invention relates to the field of biology, in particular to a method for increasing IgG1 antibody production. Background technique [0002] At present, the methods for mass production of monoclonal antibodies are mainly divided into three categories. One is in vivo method, that is, the hybridoma cells are injected into the peritoneal cavity of genotype-compatible animals to obtain monoclonal antibodies from ascites; the other is in vitro method, that is, by in vitro A large number of hybridoma cells are cultivated to produce monoclonal antibodies; the third is genetic engineering antibody technology, that is, antibody genes in monoclonal antibody hybridoma cells are cloned through genetic manipulation technology or antibody genes are directly obtained through antibody library technology, and then expressed in other expression vectors expressed in large quantities. However, hybridoma cells are not suitable for all laboratories, and mammalian cells ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/70
CPCC07K16/2863C12N15/70C07K2317/14
Inventor 赵妍淑张金华宋浩
Owner 天津大学前沿技术研究院
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