质粒DNA荧光修饰方法的研究

目的 研究一种质粒DNA荧光标记的简便方法,以便进行DNA递送的示踪.方法 质粒经 溴激活后,于不同温度下存放不同时间,再与1,10-二氨基葵烷形成氨基衍生物.氨基修饰质粒与荧光素异硫氰酸酯( FITC)反应,制备出荧光标记的质粒,纯化并收集产物,计算其标记效率,评估溴激活质粒在不同温度下存放不同时长对标记反应的影响；观察荧光标记对质粒转染效率的影响,并用激光共聚焦显微镜观察和流式细胞仪检测示踪效果.结果 实验数据显示溴活化的质粒随存放时间的延长标记效率反而下降,存放4℃较室温(25℃)的标记效率更高,提示溴活化质粒不适宜存放；而与没有标记的质粒相比,荧光标记的质粒对转染效率几乎没有影响.荧光标记的质粒应用于流式细胞仪检测和激光共聚焦显微观察都取得了较好的示踪效果.结论 本研究建立了一种荧光标记质粒DNA的简便方法,并且在基因递送的示踪实验中获得了较好的效果.

Study on the method for labeling plasmid DNA with fluorescein

Objective Conjugation of fluorescent dye onto plasmid DNA was investigated in order to monitor delivery process of plasmid DNA. Methods Plasmid was activated with bromine, stored for different time intervals at 4 ℃ or room temperature, and subsequently coupled with 1,10-diaminodeeane to prepare amine- modified plasmid DNA. Amine-modified plasmid was then reacted with isothiocyanate （FITC） for fluorescent labeling, and the labeling ratio was calculated after purification. The effect of storage conditions （time/temperature） of bromine-actived plasmid （BP） on fluorescent labeling efficacy was estimated, and the cell transfection efficiency of fluorescent plasmid-lipofectamine complex was observed. The fluorescent plasmid delivered by lipofectamine 2000 in A10 cells was observed by laser scanning confocal microscope （LSCM） and flow cytometry. Results The experimental data showed that prolonged storage time of bromine-activated DNA had a negative effect on the labeling ratio, and lower storage temperature had a positive effect on the labeling ratio. It also demonstrated that FITC modification had no effect on the transfection efficiency of plasmid-lipofectamine complex as compared with that of unlabeled plasmid-lipofectamine complex, and FITC modified plasmid had enough fluorescent intensity to monitor cell uptake with flow cytometer and sub-cellular distribution with LSCM. Conclusion A facile method for conjugating fluorescent dye onto plasmid was established in the study, and could be utilized to trace the plasmid delivery for investigating the transfection mechanism.