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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。
OBJECTIVE: To optimize the efficiency of Escherichia coli bactericidal load-carrying plasmids and to transfer plasmid-loaded bacterial exosomes to the cells to improve the delivery level of nucleic acid vaccine. Methods: Escherichia coli DH5α was transformed into plasmid Escherichia coli DH5α to prepare Escherichia coli exosomes. The ratio of bacterial cells, plasmids and membrane vesicles was optimized when plasmid pHH43 was loaded. The loading efficiency was also improved. By scanning electron microscopy and transmission electron microscopy, The morphological changes and loading efficiency of the plasmid were observed. Phagocytosis was observed by incubating plasmid-loaded bacterial slides with antigen-presenting cells, macrophages RAW264.7 and DC2.4. Results: The efficiency of E. coli bacterial load-shedding plasmids was optimized. When the proportion of bacterial cells, plasmids and membrane vesicles was 7:10:4, the efficiency was the best and the efficiency of loading DNA was over 98%. The antigen-presenting cells phagocytosed Bacterial slough plasmid, the efficiency of 100%. Conclusion: Escherichia coli can be efficiently loaded with nucleic acid vaccine and efficiently captured by antigen presenting cells, which can improve the delivery of nucleic acid vaccine and improve the immune effect.