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  

Study on the entrance of the anthrax toxin PA and LFn mediated EGFP into HeLa cells

作　　者：易绍琼[1] 于少洋[1] 于婷[1] 任声权[1] 刘树玲[1] 杨秀旭[1] 董大勇[1] 陈薇[1] 

机构地区：[1]军事医学科学院微生物流行病研究所 病原微生物生物安全国家重点实验室,北京100071

出　　处：《中华微生物学和免疫学杂志》2008年第2期158-161,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30400393）

摘　　要：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-EG-FP，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蛋白本身电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。Objective To study th,. 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 Mere amplified, respectively, and cloned into the plasmid pET- 21a （ ＋ ） one after another to construct a rocombinant plasmid pET-LFn-EGFP. The plasmid was transformed into BI21 cells to express LFn-EGFP protein under the induction of IPTG. The protein was purified by Ni chelating chromatography. After incubotion 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, Bat PA could help more LFn-EGFP molecules enter into HeLa cells.

关 键 词：致死因子 炭疽毒素保护性抗原 增强型绿色荧光蛋白 HELA细胞 炭疽杆菌 

分 类 号：R686[医药卫生—骨科学]