萤光素酶表达质粒pEE14.1-luc的构建及表达

目的:为研究10 kb以上DNA疫苗质粒的体内电穿孔递送,构建萤光素酶报告基因表达质粒p EE14.1-luc,并验证其表达。方法:从质粒p GL3-CMV中通过酶切、补平和纯化的方法获得萤光素酶基因luc,克隆入p EE14.1载体,构建重组表达质粒p EE14.1-luc,瞬时转染293T细胞,采用Western印迹、流式细胞术和免疫荧光等方法对萤光素酶基因在体外的表达情况进行验证,并运用活体成像仪检测萤光素酶基因在小鼠活体内的表达。结果:经菌液PCR鉴定和测序验证,p EE14.1-luc与预期设计完全一致;流式细胞术检测luc阳性表达率为22.41%,免疫荧光检测可见绿色荧光表达,Western印迹检测在相对分子质量为62×103处显现目的蛋白条带;同时,利用活体成像技术也检测到萤光素酶基因在小鼠活体内的表达。结论:p EE14.1-luc表达质粒构建成功,为研究DNA疫苗体内表达机制和体内电穿孔递送条件优化奠定了基础。

Construction and Expression of Recombinant Plasmid pEE14.1-luc

Objective： In order to study the delivery of DNA vaccine plasmid for over 10 kb by electroporation in vivo, we constructed the recombinant pEE14.1-luc, which can express the luciferase report gene. Methods： The luciferase gene was cut from pGL3-CMV vector, and inserted into pEE14.1-luc vector after filling in and purifica？tion. In vitro expression of the luciferase gene was identified by transient transfection to 293T cells, Western blot, flow cytometry and immunofluorescence. And in vivo expression of this gene was identified by in vivo imaging tech？nology. Results： The results indicated that pEE14.1-luc was constructed as we expected after the identification of microbial PCR and sequencing. The expression rate of luciferase was 22.41% tested by flow cytometry, and we could detect the expression of green fluorescence by immunofluorescence and a protein of 62 kD by Western blot. With in vivo imaging technology, we could also detect the expression of luciferase gene in mice in vivo. Conclu？sion： We conclude that pEE14.1-luc was successfully constructed, which lays a foundation for further studying the mechanism of in vivo expression of DNA vaccine and optimizing the in vivo conditions of delivery by electropora？tion.