GFP-HPV18 E2及其突变体融合蛋白在真核细胞内表达与定位.docVIP

GFP-HPV18 E2及其突变体融合蛋白在真核细胞内表达与定位 　　摘要：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。 　　关键词：人乳头瘤病毒18型；E2蛋白；定位 　　中图分类号： R373.9 文献标识码： A 文章编号： 1008-2409(2007)06-1188-02 　　 　　Expressionand localization of GFP-HPV18 E2 and its mutants fusion proteins in eukaryotic cells/CHEN Chun-lian, WAN Yan-ping, PENG Jun//The Affiliated Hospital of Guilin Medical College, Guilin 541001, China 　　Abstract: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 anti-GFP 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 transfectedby 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. 　　Key words:HPV18; E2; localization 　　 　　人乳头瘤病毒（human papillomavirus，HPV）18型(HPV18)属高危型，可引起重度不典型性增生??甚至恶性肿瘤[1]。HPV18早期基因区编码的E2蛋白由365个氨基酸(amino acid,aa)组成，分为N端转录活化结构域（transactivation domain,TAD）和C端DNA结合域（DNA-bind