复制子荧光素酶表达质粒pSVK-luc的构建与应用  

Construction and application of the replicon DNA recombinant plasmid pSVK-luc

作　　者：李盼[1] 张亮[2] 王宇[2] 朱晓明[2] 张巍[2] 徐元基[2] 于继云[2] 阎瑾琦[2] 

机构地区：[1]新疆大学生命科学与技术学院,新疆乌鲁木齐830046 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《细胞与分子免疫学杂志》2013年第7期765-768,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31100655)

摘　　要：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 the replicon DNA vaccine in vivo by constructing a recombinant plasmid containing luciferase reporter gene on the basis of the Semiliki forest virus （SFV） replicon vector （pSVK-luc）. Methods The luciferase gene fragment was amplified by PCR and cloned into the SFV replicon vector pSVK. The recombinant plasmid pSVK-luc was identified and screened by enzyme digestion and sequencing for the positive one. By chemical-based transfec- tion, the positive plasmid was transferred into human embryonic kidney 293T cells to observe the expression of luciferase gene by flow cytometry and immunofluorescence. By electroporation, the plasmid was delivered into BALB/c mouse quadri- ceps femoris muscles to detect the expression of target gene by in vivo imaging system. Results Sequencing revealed that the recombinant plasmid pSVK-luc we obtained was identical with the expected one. Flow cytometry and immunofluorescence showed that the expression of the luciferase gene in 293T cells and in vivo imaging system presented that the expression in the immunized mouse muscles. Conclusion A novel replicative DNA pSVK-luc has been successfully constructed, and can be expressed in 293T cells and in the muscle tissues of immunized mice, which provides a basis for future studies on the mechanism underlying the replicon DNA vaccine works in vivo and on the optimal electroporation conditions for the replicon DNA vaccine delivery in vivo.

关 键 词：荧光素酶基因 复制子载体 体内外表达 活体成像 

分 类 号：R392.33[医药卫生—免疫学] Q786[医药卫生—基础医学]