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目的制备大肠杆菌DH5α菌蜕,并用其装载核酸片段以及质粒DNA,体外转染抗原递呈细胞,探讨其作为DNA疫苗载体的可能性。方法将构建有φX174噬菌体裂解蛋白的温控型表达质粒pHH43转化入大肠杆菌DH5α中,诱导细菌裂解制备大肠杆菌DH5α菌蜕,利用制备的DH5α菌蜕装载核酸片段,并摸索出较好的装载方法用于质粒DNA的装载,然后转染巨噬细胞RAW264.7,观察转染效果。结果成功制备了大肠杆菌DH5α菌蜕,细菌的裂解效率可达97.8%,对核酸片段和质粒DNA的装载效率分别达94.89%和91.98%,装载红色荧光蛋白质粒pDSred-N1的DH5α菌蜕转染巨噬细胞RAW264.7,红色荧光蛋白的表达效率可达50%。结论大肠杆菌DH5α菌蜕可以用于装载核酸疫苗并将其靶向性地导入到抗原递呈细胞中,可获得较高的表达效率,初步验证了大肠杆菌DH5α菌蜕作为核酸疫苗载体的可行性。
Objective To prepare Escherichia coli DH5α bacterial slides, and use it to load nucleic acid fragments and plasmid DNA, and transfect antigen presenting cells in vitro to explore their potential as DNA vaccine vectors. Methods The temperature-controlled expression plasmid pHH43 with the φX174 phage lysis protein was transformed into Escherichia coli DH5α to induce bacterial lysis to produce Escherichia coli DH5α. The DH5α strain was loaded with the prepared nucleic acid fragments and the better loading method was obtained For plasmid DNA loading, and then transfected macrophages RAW264.7, observe the transfection effect. Results Bacillus subtilis DH5α was successfully prepared, the efficiency of bacterial cleavage was 97.8%, the efficiency of loading 94.89% and 91.98% of the DNA fragment and plasmid DNA respectively, and the DH5α strain carrying red fluorescent protein plasmid pDSred-N1 Macrophage RAW264.7, red fluorescent protein expression efficiency up to 50%. Conclusion Escherichia coli DH5α can be used to mount nucleic acid vaccine and can be introduced into antigen presenting cells in a targeted manner, and the high efficiency of expression can be obtained. The feasibility of using Escherichia coli DH5α as a carrier of nucleic acid vaccine .