摘要
目的构建人乳头瘤病毒16型E2(HPV16 E2)及其N端(TAD)、C端(DBD)删除突变体与增强型绿色荧光蛋白(GFP)融合蛋白的真核表达载体,观察其在Hela细胞中的表达与定位。方法HPV16 E2基因经PCR扩增后,与pMD-T载体连接,经蓝白斑筛选、PCR、双酶切鉴定及序列分析后,将E2基因亚克隆至pEGFP-C1真核表达载体中,构建荧光蛋白融合表达载体pEGFP-C1/HPV16 E2。PCR扩增TAD、DBD基因片段,将其双酶切后插入pEGFP-C1,构建荧光蛋白融合表达载体pEGFP-C1/TAD及pEGFP-C1/DBD。将3种质粒转染Hela细胞,在荧光显微镜下观察融合蛋白的表达与定位。结果重组载体经PCR、双酶切鉴定及测序证明插入片段序列正确,且在Hela细胞中能表达目的蛋白;GFP-E2融合蛋白在细胞核与细胞质中都有表达,但主要分布于细胞核;而GFP-DBD融合蛋白仅在细胞核内;GFP-TAD融合蛋白仅在细胞质中。结论构建的绿色荧光蛋白融合表达载体pEGFP-C1/HPV16 E2、pEGFP-C1/TAD及pEGFP-C1/DBD均在Hela细胞中表达,并确定了各自在细胞内的定位。
Objective To construct the eukaryotic expressing vector of human papillomavirus type（HPV16） 16 E2 protein and its N-terminus（TAD） or C-terminus deleted mutant（DBD） fused with enhanced green fluorescent protein（GFP） respectively,and investigate the fusion proteins＇ expression and localization in Hela cells. Methods HPV16 E2 gene was amplified by PCR.The product of PCR was cloned into pMD-T identified with digest enzymes and sequencing.Then HPV16 E2 gene was subcloned into eukaryotic expressing vector pEGFP-C1.HPV16 E2 TAD and DBD was amplified from pEGFP-C1/HPV16 E2 by PCR,the product was cloned into pEGFP-C1,The positive clones were screened by endonuclease restriction digestions and sequencing.The expression and localization of pEGFP-C1/HPV16 E2,pEGFP-C1/TAD and pEGFP-C1/DBD were observed under fluorescent microscope. Results The eukaryotic expressing vector encoding HPV16 E2,TAD or DBD was successfully constructed respectively,and its expression in Hela cells could be observed.Fluorescence could be observed in the whole cells transfected by pEGFP-C1/E2 and mainly in the nuclei of the cells,but only in the nuclei of the cells transfected by pEGFP-C1/DBD or only in the cytoplasma of the cells transfected by pEGFP-C1/TAD. Conclusion Eukaryotic expression vector of HPV16 E2 and TAD or DBD fused with GFP were constructed successfully respectively.Their expression could be observed in Hela cells,but their localization were different.
出处
《南华大学学报（医学版）》
2010年第1期34-37,共4页
Journal of Nanhua University(Medical Edition)
基金
湖南省科学技术厅计划项目(2009FJ3048)
