摘要
目的构建以绿色荧光蛋白(green fluorescence protein,GFP)为报告基因的重组表达质粒pEGFP-HPV16 E6。方法利用脂质体转染体外培养的Hela细胞,在活细胞状态下用荧光显微镜直接观察pEGFP-HPV16 E6融合蛋白在细胞中的分布和定位,用Western blot方法验证其蛋白的表达。结果在空载体pEGFP-C2转染组中,Hela细胞内绿色荧光呈弥散分布;重组质粒pEGFP-HPV16 E6转染组中,绿色荧光集中在细胞核中。结论pEGFP-HPV16 E6融合基因真核表达载体在真核细胞Hela中获得了表达,细胞所表达的融合蛋白具有HPV16 E6和EGFP的双重活性,可用荧光显微镜直接观察其表达情况及亚细胞定位,为HPV16 E6的功能研究提供了线索。
Objective To construct pEGFP- HPV16 E6 fusion gene in eukaryotic expression vector and to investigate its expression in Hela cells. Methods HPV16 E60RF was amplified from PCR2. 1 - HPV16E6 by PCR and inserted into plasmid pEGFP- C2. Using lipofectin method, the recombinant expression .plasmid pEGFP - HPV16 E6 was transfected into Hela cells. Conclusions The results showed that pEGFP - HPV16 E6 expressed a fusion protein with green fluorescence protein in Hela cells as defined by Western blot analysis. This fusion protein was localized in the nucleus of Hela cells as detected by fluorescence microscopy.
出处
《南华大学学报（医学版）》
2007年第3期350-354,共5页
Journal of Nanhua University(Medical Edition)
基金
湖南省科技厅专项科研基金(NO:B2004-155)
关键词
HPV16
E6
GFP表达载体
表达定位
HPV16 E6
GFP expression vector
gene expression and cellular localization
