融合蛋白表达质粒pUL54S-EGFP的构建及在AD293细胞中的表达  

Construction of UL54S-EGFP fusion protein expression vector and its expression in AD293 cells

作　　者：胡妙凤[1] 段群军[2] 陶然[2] 尚世强[2] 

机构地区：[1]浙江大学医学院附属儿童医院麻醉科 [2]浙江大学医学院附属儿童医院中心实验室

出　　处：《浙江医学》2008年第8期832-834,837,共4页Zhejiang Medical Journal

基　　金：国家自然科学基金(30571663)

摘　　要：目的探讨构建人巨细胞病毒(HCMV)截短UL54(UL54S)基因融合增强型绿色荧光蛋白(EGFP)表达质粒pUL54S-EGFP转染人胚肾293细胞,瞬时表达UL54S和EGFP的可行性。方法采用PCR法扩增UL54s基因并将其克隆入真核表达载体pEGFP-N1中,采用脂质体法转染AD293细胞后通过荧光显微镜和流式细胞仪分析EGFP的表达情况。结果重组质粒pUL54S-EGFP转染AD293细胞12h后即可见到荧光蛋白的表达,至48h表达达高峰;转染48hpUL54S-EGFP组平均转染率为47%,平均荧光强度为547。结论重组质粒pUL54S-EGFP可在人胚肾293细胞内瞬时表达UL54S和EGFP融合蛋白,EGFP的表达可报告UL54基因的表达,质粒pUL54S-EGFP构建成功。Objective To construct plasmid pUL54S-EGFP （truncated region of HCMV UL54 and enhanced green fluorescent protein） , and to express the fusion protein in AD293 cells. Methods The fusion protein expression vector pUL54S-EGFP was constructed by inserting the 784bp Hind Ⅲ -EcoR Ⅰ fragment （UL54S） into the same sites of pEGFP-N1, and the plasmid was transfected in AD293 cells with Lipofectamine. The expression of fusion gene UL54S-EGFP was analyzed by fluorescence microscopy and flow cytometer. Results Fluorescent signals were detected in pUL54S-EGFP group at 12h and reached a peak at 48h after transfection. Flow cytometry demonstrated that the transfection efficiency was 47% and mean fluorescence intensity was 547 for pUL54S-EGFP group at 48h after transfection. Conclusion The recombinant plasmid carrying EGFP and truncated region of HCMV UL54 gene is constructed successfully, and it can express UL54S-EGFP in AD293 cells.

关 键 词：人巨细胞病毒 UL54基因 增强型绿色荧光蛋白 

分 类 号：R346[医药卫生—基础医学]