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CHO cell strain modified based on CRISPR/Cas9 gene editing and preparation method thereof

A gene editing and cell line technology, applied in the field of gene editing, can solve the problems of high price and complicated design, and achieve the effects of strong anti-apoptotic ability, high catalytic disulfide bond efficiency, and high protein expression quality.

Pending Publication Date: 2019-11-05
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two methods can further expand the scope of gene editing, but the design is complicated and the price is relatively expensive, which limits their large-scale application to a certain extent.

Method used

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  • CHO cell strain modified based on CRISPR/Cas9 gene editing and preparation method thereof
  • CHO cell strain modified based on CRISPR/Cas9 gene editing and preparation method thereof
  • CHO cell strain modified based on CRISPR/Cas9 gene editing and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Design, construction and functional verification of sgRNA-Cas9 and expression cassette based on CHO-K1 cells

[0031] Reagents and kits: Premix Taq DNA polymerase, Pyrobest DNA polymerase, restriction endonucleases (Hind III, BamH I), DNA A-Tailing Kit, pMDTM19-T Vector Cloning Kit, DNAmarker were purchased from Bao Biological Engineering Co., Ltd. (Dalian). Endonuclease Bbs I and T4 DNA ligase were purchased from NEB. Plasmid Extraction Kit, Blood / Cell, Tissue Genomic DNA Extraction Kit, EasyGene Rapid Recombination Cloning Kit, DNA Gel Recovery Kit and UniversalDNA Purification and Recovery Kit were purchased from Tiangen. Lipofectamine 2000 was purchased from Thermo Fisher Scientific. Tryptone and yeast extract were purchased from Oxoid Company (UK). Ampicillin was purchased from Sangon Bioengineering Co., Ltd. (Shanghai). F-12 and Opti-MEM medium, trypsin were purchased from Gibco. Penicillin-streptomycin (100×) was purchased from Solarbio. FBS was p...

Embodiment 2

[0078] Example 2. Establishment of monoclonal stable cell lines overexpressing HsQSOX1b and Survivin

[0079] Reagents and kits: PMSF and BCAProtein Assay kit were purchased from Shanghai Sangong. HRP-conjogated goat anti-mouse IgG (H+L), HRP-conjogated goat anti rabbit, Anti-βactin antibody and Anti-QSOX1 antibody were purchased from Proteintech. Pro-light HRPchemiluminescent kit was purchased in Tiangen, Beijing. ER-Tracker Red and Hoechst 33342 dye were purchased from Thermo Fisher Scientific. Other biochemical reagents belong to domestic conventional analytical reagents.

[0080] Cells, strains and plasmids: recombinant plasmids sgRNA1-QSOX1-pX458(Q1), sgRNA2-QSOX1-pX458(Q2), sgRNA1-BIRC5-pX458(B1) and sgRNA2-BIRC5-pX458(B2) were constructed for the above stages of the present invention . E. coli DH5α (Invirogen, USA) was used as a plasmid amplification strain; cell line CHO-K1 cells (Cell Bank of Chinese Academy of Sciences).

[0081] Reagent preparation:

[0082] 5...

Embodiment 3

[0097] Example 3. Preliminary study on the quality control of intracellular protein in monoclonal strains overexpressing HsQSOX1b and Survivin

[0098] Reagents and kits: PrimeScript TM RT Master Mix, Premix Ex Taq and restriction endonucleases (Hind III, BamH I) were purchased from TaKaRa. Annexin V-PE apoptosis detection kit and GSHand GSSG Assay Kit were purchased from Beyotime. Gaussia Luciferase Assay Kit was purchased from NEB. PI Staining Solution was purchased from Yeasen. Other biochemical reagents belong to domestic conventional analytical reagents.

[0099] Cells and plasmids: Cells: monoclonal strain EC#1-C1 stably expressing HsQSOX1b-KDEL and monoclonal strain EC#2-B6 stably expressing HsQSOX1b-KDEL and Survivin; plasmids: pGluc-Basic, and pCDNA3.1.

[0100] Reagent preparation:

[0101] Preparation of 5mg / mL MTT stock solution: Accurately weigh 250mg MTT and dissolve it in 50mL of PBS, then filter and sterilize with a 0.22μm sterile filter membrane, aliquo...

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Abstract

The invention provides a CHO cell strain modified based on CRISPR / Cas9 gene editing and a preparation method thereof. Sulfhydryl oxidase (HsQSOX1b) related to disulfide bond folding and survivin related to apoptosis are selected as target genes and designed as heterogenous expression cassettes EC#1 and EC#2, and a novel CRISPR / Cas9 gene editing technology is used for integrating the heterogenous expression cassettes into a specific genetic locus of a CHO cell. According to the CHO cell strain modified based on CRISPR / Cas9 gene editing and the preparation method thereof, the CRISPR / Cas9 gene editing technology is used, human HsQSOX1b and survivin genes accurately edit a CHO-K1 cell, a rapid and efficient host cell modification technology platform is established, a monoclonal cell strain with high anti-apoptosis ability and high disulfide bond catalytic efficiency and protein expression quality is obtained, and a theoretical and technical support is provided for modification of an efficient antibody cell strain which meet the requirements of industrial production.

Description

technical field [0001] The present invention relates to the technical field of gene editing, and more specifically relates to a CRISPR / Cas9 gene editing technology-based transformation of CHO cells with HsQSOX1b and Survivin as target genes. Background technique [0002] In recent years, therapeutic recombinant proteins represented by antibodies have become the leading products in the biopharmaceutical industry, and there is a huge clinical demand. Since the approval of the first recombinant protein human tissue plasminogen activator (tPA) produced in CHO cells in 1986, CHO cells have become the most important expression host for exogenous protein production. However, in the industrial production of recombinant proteins, the quality and quantity of target protein expression are low, and the resistance to adverse environments is weak. Bottlenecks have always affected the economic benefits and clinical applications of recombinant proteins. With the sequencing of the genome of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85
CPCC12N5/0682C12N15/85C12N9/0051C12Y108/03002C07K14/47C12N2510/00
Inventor 马兴元王文鹏郑文云胡发彪胡凤枝何秀娟张晨
Owner EAST CHINA UNIV OF SCI & TECH
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