GFP-HPV18 E2及其突变体融合蛋白在真核细胞内的表达与定位

目的:研究HPV18 E2及其N端(TAD)、C端(DBD)与GFP融合蛋白在巨噬细胞(MΦ)内的表达与定位。方法:将真核表达载体pEGFP-C1/E2、pEGFP-C1/TAD和pEGFP-C1/DBD及pEGFP-C1分别转染MΦ,48h后用倒置荧光显微镜观察荧光情况,以确定GFP及融合蛋白的表达;以抗GFP抗体为一抗作Western blot检测相应蛋白质的表达。结果:在荧光显微镜下,4种质粒转染的细胞,pEGFP-C1组细胞内均有荧光;pEGFP-C1/E2组荧光主要在细胞核,但细胞浆也有;pEGFP-C1/DBD仅在细胞核有荧光,pEGFP-C1/TAD仅在细胞浆有荧光。Western blot检测到GFP-HPV18 E2及其突变体融合蛋白在细胞内的表达。结论:GFP-E2及其突变体融合蛋白均能在巨噬细胞内表达,且GFP-E2主要定位于细胞核,GFP-DBD定位于细胞核内,GFP-TAD定位于细胞浆。

Expression and localization of GFP-HPV18 E2 and its mutants fusion proteins in eukaryotic cells

Objective:To study expression and localization of GFP-HPV18 E2 and its TAD or DBD fusion proteins in macrophages.Methods:The plasmid of pEGFP-C1/ E2,pEGFP-C1/TAD,pEGFP-C1/DBD or pEGFP-C1 was transfected into macrophages,respectively.Their expression and localization were observed with fluorescent microscope.Western blot was used to analyze GFP or its fusion proteins by the antiGFP antibody as the first antibody.Results:Fluorescence could be observed in the whole cells transfected by pEGFP-C1,mainly in the nuclei of the cells transfected by pEGFP-C1/E2,but only in the nuclei of the cells transfected by pEGFP-C1/DBD while only in the cytoplasma of the cells transfected by pEGFP-C1/TAD.Western blot showed that the expression of GFP,GFP-E2,GFP-DBD,GFP-TAD fusion proteins.Conclusion:GFP-HPV18 E2 and its mutants could express in macrophages.GFP-E2 fusion protein locate mainly in nuclei,while GFP-DBD fusion protein locate completely in nuclei and GFP-TAD fusion protein locate completely in cytoplasma.