Codon optimization improves the expression of nucleoprotein of rabies virus CTN-1 strain in E. coli
10.3760/cma.j.cn112309-20201016-00478
- VernacularTitle:密码子优化提高狂犬病病毒CTN-1株核蛋白在大肠埃希菌中的表达
- Author:
Yuanxue REN
;
Xin GAO
;
Qian LIU
;
Xuexin LU
;
Wuyang ZHU
- From:
Chinese Journal of Microbiology and Immunology
2021;41(5):333-337
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To obtain the high-efficiency expression of the biological active rabies virus nucleoprotein in the prokaryotic expression system.Methods:This experiment uses codon optimization technology to re-encode the nucleoprotein gene of rabies virus CTN-1 strain, artificially synthesize the full-length gene and clone it into pET-43.1a prokaryotic expression vector, induced expression in BL21 (DE3) strain of Escherichia coli( E. coli), and used Western blot to detect its reactogenicity. Results:The results showed that after induction, SDS-PAGE electrophoresis analysis showed that an obvious expression band appeared at a molecular weight of 50×10 3, which was consistent with the expected protein band size. Among them, the E. coli concentration A600 is about 0.5, and the expression yield is the highest (about 32.3%) when induced at 37℃ for 5 h. Nucleoprotein expression product is mainly inclusion body when it is expressed in large quantities. After purification by Ni 2+ chelating chromatography, the purity of the target protein can reach over 95%. The purified product was identified by Western blot and positively reacted with the sera of mice immunized with rabies vaccine, indicating that the prokaryotic expression of the CTN-1 strain nucleoprotein has biological activity. Conclusions:This experiment successfully established a high-efficiency expression method for the nucleoprotein of the CTN-1 strain in the prokaryotic expression system, and obtained high-purity target protein, which provides a basis for further clinical diagnosis and preparation of new vaccines.
