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目的为研究复制子DNA疫苗在生物活体内的表达情况,构建含有荧光素酶报告基因的复制子表达质粒pSVK-luc。方法 PCR扩增荧光素酶报告基因,克隆入DNA复制子载体pSVK,酶切鉴定和测序分析筛选阳性重组质粒pSVK-luc;化学法转染人胚肾293T细胞,流式细胞术和免疫荧光法检测荧光素酶基因在293T细胞中的表达;利用基因电穿孔导入仪递送重组质粒至BALB/c小鼠股四头肌,活体成像仪动态观测荧光素酶基因在小鼠体内的表达。结果 pSVK-luc经相应酶切和测序鉴定,与预期设计完全一致。流式细胞术和免疫荧光检测均可见荧光素酶基因表达;同时活体成像仪也能检测到该基因在小鼠体内的表达。结论 pSVK-luc表达质粒的构建成功将为复制子DNA疫苗体内作用机制研究和体内电穿孔递送条件的优化奠定实验基础。
Objective To study the expression of replicon DNA vaccine in vivo and to construct a replicon expression plasmid pSVK-luc containing luciferase reporter gene. Methods The luciferase reporter gene was amplified by PCR and cloned into the DNA replication vector pSVK. The recombinant plasmid pSVK-luc was screened by restriction enzyme analysis and sequencing analysis. 293T cells were transfected with human embryonic kidney 293T cells by flow cytometry and immunofluorescence The expression of luciferase gene was detected in 293T cells. The recombinant plasmids were delivered to quadriceps femoris of BALB / c mice by electroporation electroporation, and luciferase gene expression in mice was observed by live imager. Results pSVK-luc was identified by restriction enzyme digestion and sequencing, which was exactly the same as the expected design. The expression of luciferase gene was observed by flow cytometry and immunofluorescence assay. Meanwhile, the gene expression in mice was also detected by the live imager. Conclusion The successful construction of pSVK-luc expression plasmid will lay the experimental foundation for the study of the mechanism of action of the replicon DNA vaccine in vivo and the optimization of electroporation delivery conditions in vivo.