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Dnmt3b gene deficient type CHO (Chinese hamster ovary) cell line, preparation method and application thereof and recombinant protein expression system

A gene defect and expression system technology, applied in the field of genetic engineering, can solve problems such as low expression level, unsatisfactory expression stability of target protein, promoter DNA methylation, etc., to improve expression level, improve expression stability, overcome low expression effect

Pending Publication Date: 2019-09-20
XINXIANG MEDICAL UNIV
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Problems solved by technology

However, when the system was further used for expression stability verification, it was found that the system still had the problem of unsatisfactory expression stability of the target protein, and promoter DNA methylation still occurred to a certain extent; according to long-term expression in production without pressurized screening In the absence of G418 screening pressure, the expression maintenance rate of the recombinant protein has not reached 70% after 30 passages, and the expression level is still low

Method used

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  • Dnmt3b gene deficient type CHO (Chinese hamster ovary) cell line, preparation method and application thereof and recombinant protein expression system
  • Dnmt3b gene deficient type CHO (Chinese hamster ovary) cell line, preparation method and application thereof and recombinant protein expression system
  • Dnmt3b gene deficient type CHO (Chinese hamster ovary) cell line, preparation method and application thereof and recombinant protein expression system

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Experimental program
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Embodiment 1

[0050] The preparation method of the DNA methyltransferase Dnmt3b gene-deficient CHO cell line in this embodiment includes the following steps:

[0051] 1. Determine the target site for candidate genes

[0052] 1) Amplify the partial sequence of DNA methyltransferase gene Dnmt3b gene:

[0053] According to NCBI GenBank Chinese hamster DNA methyltransferase gene Dnmt3b genome sequence (No. NW_006879210) design amplification primers:

[0054] Dnmt3b-Ex1PCR-L: 5'-GTGCCCCCATTTCTCCTACT-3' (as shown in SEQ ID NO.3);

[0055] Dnmt3b-Ex1PCR-R: 5'-AGACCCAATGTGCTGGTCTC-3' (as shown in SEQ ID NO.4);

[0056] Perform PCR amplification of Dnmt3b gene fragments, and clone and sequence the PCR amplified fragments to verify the accuracy of the sequence. The verified amplified sequence is shown in SEQ ID NO.2.

[0057] 2) Determine the sequence of the sgRNA targeting site:

[0058] The sgRNA targeting site sequence of Dnmt3b gene assisted by online tool (http: / / crispr.mit.edu / ) is as follows:

[0059] D3b-...

Embodiment 2

[0091] Expression analysis of recombinant antibody (adalimumab) in recombinant CHO cells

[0092] To further test the effect of Dnmt3b-deficient CHO cells on the stability of recombinant protein (antibody) expression, based on plasmid pWTY-02, a eukaryotic expression vector driven by CMV promoter to express adalimumab was constructed. The constructed expression vector plasmids were transfected into Dnmt3b-deficient (3b-7) and normal CHO cells (CHO-K1) respectively. The transfected cells were cultured in a medium containing G418 (800 μg / mL) for 15 days to select a stable transfected cell pool, and the recombinant CHO cells were subcultured to 30 passages every 3 days. Subsequently, the recombinant CHO cells were cultured in a 125 mL culture shake flask with 30 mL protein-free, serum-free, and chemically determined CD CHO medium (Life Technologies, the medium containing 8mM L-glutamine) for 6 days to reach the number of cells Reach 1.5×10 7 , Collect the cells every day and use th...

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Abstract

The invention relates to a DNA transmethylase Dnmt3b gene deficient type CHO (Chinese hamster ovary) cell line, a preparation method and application thereof and a recombinant protein expression system, and belongs to the technical field of genetic engineering. CHO cell Dnmt3b gene is knocked off by CRISPR / Cas9 gene editing technique so as to obtain the Dnmt3b gene deficient type CHO cell line is attained; the Dnmt3b gene deficient type CHO cell line can evidently increase the expression level and expression stability of a target gene in CHO cells, and the problem can be solved that existing CHO cell expression systems have low expression level and expression instability; upon expression of recombinant Adalimumab with the cell line herein, it is discovered that the expression level of the recombinant Adalimumab is increased evidently. Therefore, the cell line of the invention is widely applicable to the expression of target proteins.

Description

Technical field [0001] The invention relates to a Dnmt3b gene-deficient CHO cell line, a preparation method and application thereof, a recombinant protein expression system, and belongs to the technical field of genetic engineering. Background technique [0002] Because of its outstanding features such as the ability to assemble and fold recombinant proteins and post-translational modifications, mammalian cells, especially Chinese hamster ovary (CHO) cells, are currently often used for recombinant drug protein expression and production. However, in the long-term culture and production of recombinant CHO cells at this stage, there are phenomena such as low expression level, unstable expression and even significant decrease in expression level. For this reason, in mammalian cells, how to improve the expression level and expression stability has become a major problem to be solved urgently for large-scale production of recombinant proteins. Currently, due to the lack of recombinant...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/90C12N9/22C12N15/85C07K16/24
CPCC12N9/1007C12Y201/01037C12N15/907C12N9/22C12N15/85C07K16/241C12N2810/10C12N2800/107C07K2317/14C12N15/1137C12N2310/20C12N15/90C12N2800/80
Inventor 贾岩龙王天云路江涛郭潇倪天军肖梦珂邱乐乐林艳赵春澎
Owner XINXIANG MEDICAL UNIV
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