CAJ | 학술논문

目的研究炭疽毒素保护性抗原(protective antigen,PA)和致死因子(lethal factor,LF)N端254个氨基酸(LFn)在辅助增强型绿包荧光蛋白(EGFP)进入细胞中的作用.方法分别扩增炭疽毒素的基因片段和EGFP的基因全长,将两片段先后克隆至pET-21a(+),构建成重组表达质粒pET-LFn-EGFP,在大肠杆菌中诱导表达,并对融合蛋白LFn-EGFP进行纯化.利用荧光共聚焦显微镜和流式细胞术研究LFn-EGFP融合蛋白进入细胞的情况.结果获得较高纯度的融合蛋白LFn-EGFP,纯度可达90%以上,体外实验显示该融合蛋白保留了LFn与PA结合的活性,并且能够进入到HeLa细胞中.结论 LFn-EGFP蛋白本身也会以未知的机制进入细胞,在PA辅助时,LFn-EGFP进入细胞的效率会有所增加.
목적 연구탄저독소보호성항원(protective antigen,PA)화치사인자(lethal factor,LF)N단254개안기산(LFn)재보조증강형록포형광단백(EGFP)진입세포중적작용.방법 분별확증탄저독소적기인편단화EGFP적기인전장,장량편단선후극륭지pET-21a(+),구건성중조표체질립pET-LFn-EGFP,재대장간균중유도표체,병대융합단백LFn-EGFP진행순화.이용형광공취초현미경화류식세포술연구LFn-EGFP융합단백진입세포적정황.결과 획득교고순도적융합단백LFn-EGFP,순도가체90%이상,체외실험현시해융합단백보류료LFn여PA결합적활성,병차능구진입도HeLa세포중.결론 LFn-EGFP단백본신야회이미지적궤제진입세포,재PA보조시,LFn-EGFP진입세포적효솔회유소증가.
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.