大肠杆菌菌蜕装载质粒DNA实验方法的优化

目的:优化大肠杆菌菌蜕装载质粒的效率,并将装载质粒的菌蜕转染抗原提呈细胞,以提高核酸疫苗的递送水平。方法:将质粒pHH43转化大肠杆菌DH5α,制备大肠杆菌菌蜕;优化菌蜕装载质粒时菌蜕、质粒和膜囊的比例,获得更高的装载效率,通过扫描及透射电镜、流式细胞术观察其形态变化及装载效率;将装载质粒的菌蜕与抗原提呈细胞——巨噬细胞RAW264.7和树突状细胞DC2.4共孵育,观察吞噬效果。结果:优化了大肠杆菌菌蜕装载质粒的效率,当菌蜕、质粒、膜囊的比例为7∶10∶4时效率达到最佳,装载DNA效率达98%以上;抗原提呈细胞吞噬装载了质粒的菌蜕,效率达100%。结论:大肠杆菌菌蜕可高效装载核酸疫苗,且高效被抗原提呈细胞捕获,有助于提高核酸疫苗的递送和免疫效果的提高。

Optimization of Plasmid DNA Loading Procedure for Escherichia co-li Bacterial Ghosts Mediated Gene Delivery

Objective： To optimize plasmid DNA loading procedure of E.coli bacterial ghosts（BG）, and to trans-fect the resulting BG into antigen presenting cells（APC） to improve the delivery efficiency of DNA vaccine. Methods： Plasmid pHH43 was transformed into E.coli and the E.coli BG was prepared by lysis E protein in a temperature controlled manner. The plasmid DNA were loaded into E.coli BG and for higher efficiency, the ratios of BG, plasmids,and membrane envelope were optimized during the DNA loading. Then BG with plasmid were ob-served and detected by transmission electron microscope, scanning electron microscope and FACS. BG loaded with plasmids were incubated with APC human macrophage cells RAW264.7 and human dendritic cells DC4.2. Re-sults： The efficiency of E.coli loading plasmid was improved to more than 98% when the ratio of BG, plasmids, and membrane envelope was 7∶10∶4. The BG loaded with plasmid DNA could be observed in almost all APC. Conclusion： The efficiencies of E.coli BG loading DNA plasmid and subsequent transfecting APC were high so that DNA vaccine delivery can be accomplished.