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Method for screening glutamine synthetase-deficient HEK293 cell line

A glutamine and synthetase technology, applied in the biological field, can solve the problems of no screening ability, only 26% positive rate of cell screening, and inability to screen high gene copy number cell clones, etc., to simplify the culture process and have good clinical application prospects and commercial value, and the effect of improving the expression of recombinant protein

Active Publication Date: 2018-04-10
EUREKA BIOTECH LTD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the Glutamine synthetase / Methioninesulfoximine (GS / MSX) screening system widely used on the CHO platform cannot be used because HKE293 highly expresses the GS gene and is not sensitive to glutamine.
Previous studies have found that the GS activity of HEK293 is about 4.8 times that of CHO cells. The MSX concentration below 500 μM has basically no screening ability for HEK293 cells. Even at a high MSX concentration of 1000 μM, the positive rate of cell selection is only 26%, and it cannot be screened. Screen cell clones with high gene copy number and high protein expression by further increasing the concentration of MSX

Method used

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  • Method for screening glutamine synthetase-deficient HEK293 cell line
  • Method for screening glutamine synthetase-deficient HEK293 cell line
  • Method for screening glutamine synthetase-deficient HEK293 cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Design GS gene sgRNA and construct CRISP / Cas9 plasmid

[0088] (1) Design GS gene sgRNA sequence

[0089] By comprehensively comparing the prediction results of various online tools, analyzing the number of mismatches, GC content, out-of-frame score and other conditions, in the third to eighth exon interval of the GS gene coding region ( SEQ ID NO.1) designed a total of 81 sgRNA sequences (see Table 1 for specific sequences).

[0090] Table 1 sgRNA sequence of GS gene coding region

[0091]

[0092]

[0093]

[0094] (2) Construction of pX330-GS-sgRNA (E3#01-E8#13) vector

[0095] Add BbsI cohesive end to the 5' end of the sgRNA sequence in Table 1, design the reverse complementary sequence, and synthesize corresponding primers (Table 2). The synthesized sgRNA forward and reverse complementary primers were dissolved in T4 polynucleotide kinase solution at a final concentration of 0.5 μM, and catalyzed by T4 polynucleotide kinase (T4 Polynucleotide K...

Embodiment 2

[0100] Example 2: Construction of pShCMV-EGx-GSE(3-8)-xFP plasmid

[0101] (1) Construction of pShCMV-EGx-MCS-xFP plasmid.

[0102] With plasmid pCMV(PacI)-MCS-IRES-EGFP (plasmid map as Figure 12 Shown) is the template PCR amplification enhanced green fluorescent protein (Enhanced Green Fluorescent Protein, EGFP) gene fragment. The primers for EGFPfrag1 were 5'acagATCGATgccaccATGGTGAGCAAGGGCGA G and 5'CTGggatccgaattcAGTGGTTGTCGGGCAGCAG; the primers for EGFPfrag2 were 5'TCACctcgagGCAAGCTGACCCTGAAGTTC and 5'CTACTGagatctTTACTTGTACAGCTCGTCCATG. The PCR reaction uses KAPAHiFiDNA Polymerase, the annealing temperature is 58°C, and the extension is 15s. The recovered EGFP1 fragment and pShCMV-MCS (MCS sequence is shown in SEQ ID NO.83, and the plasmid map is shown in Figure 13 (shown) the plasmid was digested with ClaI and BamHI, gel recovery and purification, T4 DNA ligase ligation, DH5α competent cell transformation and small amount of plasmid DNA preparation and identification...

Embodiment 3

[0107] Example 3: Rapid sgRNA efficiency detection

[0108] A total of 16 samples of pX330-GS-sgRNA (E3#01) to (E3#16) and pShCMV-EGx-GSE3-xFP plasmid; pX330-GS-sgRNA (E4#01) to (E4#10) and pShCMV- A total of 10 samples of EG x-GSE4-xFP plasmids (where E4#2-1 and E4#2-2 were co-transfected in equal mass ratios); pX330-GS-sgRNA (E5#01) to (E3#15) and pShCMV -15 samples of EGx-GSE5-xFP plasmid; pX330-GS-sgRNA(E6#01) to (E6#10) and pX330-GS-sgRNA(E7#01) to (E7#16) with pShCMV-EGx- A total of 26 samples of GSE(6&7)-xFP plasmid; a total of 13 samples of pX330-GS-sgRNA(E8#01) to (E8#13) and pShCMV-EGx-GSE8-xFP plasmid were co-transfected by calcium phosphate coprecipitation method HEK293 cells. The pX330 plasmid that does not contain the sgRNA sequence was mixed with pShCMV-EGx-GSE3-xFP, pShCMV-EGx-GSE4-xFP, pShCMV-EGx-GSE5-xFP, pShCMV-EGx-GSE(6&7)-xFP, pShCMV-EGx-GSE8 -xFP co-transfection with five plasmids served as a negative control. The experimental steps are as follows:

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Abstract

The present invention belongs to the field of biotechnology, and discloses glutamine synthetase-deficient cell line HEK293-GS- / -which is based on HEK293 cells, can be stably passaged, is adapted to suspension culture, can be applied to the expression of recombinant proteins, and is constructed by using of a CRISPR / Cas9 system. The glutamine synthetase-deficient cell line HEK293-GS- / -can screen engineered cell strains expressing various recombinant proteins through a GS / MSX screening system. The glutamine synthetase-deficient cell line HEK293-GS- / -can stably express the target proteins after multiple passages; constructed cells can be adapted to most of commercial serum-free media; the screened cells themselves express high levels of glutamine synthetase, and the upstream culture process isgreatly simplified. The glutamine synthetase-deficient cell line HEK293-GS- / -can be applied to molecular biology, cell biology and other research fields, and is also suitable for constructing engineering cells used in the field of biopharmaceuticals.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for screening glutamine synthetase (GS)-deficient HEK293 cell lines and a method for screening recombinant protein-expressing cell lines based on the glutamine synthetase-deficient HEK293 cell lines. Background technique [0002] Protein modifications (post-translational modifications, PTMs) play a crucial role in protein activity, stability and immunogenicity, and directly affect the efficacy, half-life and drug resistance of recombinant protein drugs. Future drug upgrades and new drug development must focus on the similarity between PTMs and human proteins. The use of human cells to produce recombinant proteins is a shortcut to resolve the variability of PTMs. [0003] HEK293 cells are derived from human embryonic kidney cells and immortalized by transfection of adenovirus 5 gene. The transfected HEK293 cell line genome carries the adenovirus 5E1 region, and expresses tw...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/90C12N9/22C12N5/10C12Q1/02
CPCC12N5/0686C12N9/22C12N15/113C12N15/907C12N2310/10C12N2510/00G01N33/5044
Inventor 薛博夫马墨杨银辉白孟飞陈莉胡雯钟宇
Owner EUREKA BIOTECH LTD
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