Optimizing the secretory expression of SARS-CoV-2 S-EABR in 293T cells
10.3760/cma.j.cn112866-20240720-00110
- VernacularTitle:优化SARS-CoV-2 S-EABR在293T细胞中的分泌性表达
- Author:
Surui JIANG
1
;
Tongyao MAO
;
Peng ZHANG
;
Zhaojun DUAN
Author Information
1. 甘肃中医药大学公共卫生学院，兰州 730000
- Keywords:
SARS-CoV-2;
S-EABR;
Promoter;
PolyA signals;
Signal peptide;
SRP
- From:
Chinese Journal of Experimental and Clinical Virology
2024;38(5):489-496
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To increase secretory expression of SARS-CoV-2 S-EABR protein in 293T cell line by optimizing promoter, PolyA signals, signal peptide and terminal amino acids of intracellular sequences.Methods:First, four PCDNA3.1 (-) eukaryotic vector plasmids (Mb, MS, Ab, AS) containing different combinations of elements (promoter and PolyA signals) were constructed, and the S-EABR-1 target sequence optimized according to human codons was inserted. 293T-cells were transiently transfected. After 48 hours, cell culture supernatants and cell lysates were collected, and the expression level of S protein in supernatant was detected by Western blotting and ELISA. Then, the vector with the best expression element combination was selected, and the target sequences of S-EABR-1 and S-EABR-2 (4 amino acids-HSLP were added to the tail of S-EABR-1) were inserted to compare the expression level of S protein in the supernatant. Finally, based on the combination of the above elements with the best expression effect and the insertion of the target sequence, five vector plasmids (tPA, AZ, IFNα2, HSA, GLUC) were constructed to replace the original signal peptide of SARS-CoV-2 S protein, and the expression level of S protein in the supernatant was compared. At the same time, a computer was used to simulate the molecular docking of the SRP54 subunit and the signal peptide nucleic acid sequence, and the Docking Score was used as the docking evaluation criterion to predict the binding of the two.Results:In 293T cells, the Ab combination vector secreted the highest level of S-EABR, and the yield increased by 125% compared with Mb. Based on the Ab combination vector, the level of S-EABR-2 sequence expression and secretion of S-EABR increased by about 50% compared with S-EABR-1. After further replacement with the HSA signal peptide, the level of S-EABR expression and secretion increased by about 83% compared with the original signal peptide of the S protein. In addition, computer simulation result showed that the docking score between HSA and SRP54 subunit was the highest, at 1 505.861.Conclusions:The secretory expression of codon-optimized S-EABR in 293T cells can be further improved by optimizing eukaryotic expression elements (promoter, terminator and signal peptide) and intracellular sequences. The calculated simulated docking score of the affinity between the signal peptide and the SRP54 subunit is basically consistent with the secreted expression level of S-EABR also provides a design idea and screening strategy for subsequent screening of signal peptides to improve the secreted expression of the target gene.
