Study on the entrance of the anthrax toxin PA and LFn mediated EGFP into HeLa cells
- VernacularTitle:炭疽毒素保护性抗原和LFn介导的增强型绿色荧光蛋白进入HeLa细胞的研究
- Author:
Shaoqiong YI
;
Shaoyang YU
;
Ting YU
;
Shengquan REN
;
Shuling LIU
;
Xiuxu YANG
;
Dayong DONG
;
Wei CHEN
- Publication Type:Journal Article
- Keywords:
Lethal factor;
LFn-EGFP fusion protein
- From:
Chinese Journal of Microbiology and Immunology
2008;28(2):158-161
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the role of protective antigen(PA)and N-terminal segment of lethal factor (LFn)in the entrance of EGFP(enhanced green fluorescent protein)into HeLa cells. Methods The DNA fragments encoding LFn and EGFP were amplified,respectively,and cloned into the plasmid pET-21 a(+)one after another to construct a recombinant plasmid pET-LFn-EGFP. The plasmid was txansformed into BL21 cells to express LFn-EGFP protein under the induction of IPTG. The protein was purified by Ni chelating chromatography. After incubation with LFn-EGFP in the presence of PA or not, the HeLa cells were analyzed by flow cytometry or laser confocal microscopy. Results The fusion protein LFn-EGFP was purified by over 90% homogeneity and retained the ability of LF to bind with PA when incubated with J774A.1 macrophage cells,and could get into HeLa cells. Conclusion The LFn-EGFP could enter the HeLa cells in a PA independent pathway. But PA could help more LFn-EGFP molecules enter into HeLa cells.
