真核表达载体pEGFP-N1-VP3的构建及在SGC-7901细胞中的表达和定位  

Construction of eukaryotic pEGFP-N-VP3 expressed vector and its expression and localization in the gastric cancer SGC-7901 cells

作　　者：刘少宁[1] 祖毅 向廷秀[3] 王丕龙[1] 王丽娟[1] 曹红丹[1] 吕琳[1] 

机构地区：[1]重庆医科大学附属第一医院消化内科,重庆400016 [2]重庆市第八人民医院,重庆400016 [3]重庆医科大学附属第一医院实验研究中心,重庆400016

出　　处：《生物学杂志》2009年第1期15-18,30,共5页Journal of Biology

基　　金：国家自然科学基金青年基金(30500234);重庆市卫生局自然科学基金(05-2-175)

摘　　要：构建真核表达载体pEGFP-N1-VP3并稳定转染人胃癌细胞SGC-7901,观察EGFP-VP3融合蛋白在肿瘤细胞中的分布和亚细胞定位,探讨凋亡素诱导肿瘤细胞凋亡的机制。用PCR技术扩增出(凋亡素)VP3基因片段,克隆至载体pEGFP-N1,鉴定无误后,将构建的重组质粒pEGFP-N1-VP3经脂质体介导转染SGC-7901细胞,在荧光显微镜和激光扫描共聚焦显微镜下观察凋亡素在肿瘤细胞中的分布、亚细胞定位。用AO/EB荧光染色法检测其在体外诱导肿瘤细胞凋亡的效应。经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入载体pEGFP-N1,稳定转染细胞中EGFP-VP3在肿瘤细胞中得以高表达,转染后逐渐从细胞质迁移至细胞核,最后定位于细胞核内。AO/EB荧光染色观察到大量细胞凋亡。成功构建真核表达载体pEGFP-N1-VP3,并成功培养出表达绿色荧光蛋白和凋亡素的SGC-7901稳定细胞株。EGFP-VP3融合蛋白在肿瘤细胞中具有核定位效应,并诱导肿瘤细胞凋亡。The VP3 gene fragment was amplified by PCR, and inserted into expression vector pEGFP-N1. After being digested, the recombinant plasmid was then transferred into SGC-7901 cells by liposome. The distribution and subcellular location of fusion protein EGFP-VP3 in tumor ceils were observed under fluorescence microscopy and laser scanning confocal microscope. The VP3 induced apoptosis of tumor cells in vitro was tested by AO/EB assay. The result showed that the target gene was cloned into recombinant vector. High expression of EGFP-VP3 was detected in the transfeeted tumor cells, and EGFP-VP3 moved from cytoplasm to nucleus gradually, finally accumulated in nucleus. Apoptosis of induced tumor cells was observed after transfection by AO/EB assay. The eukaryotic expression vector pEGFP-N1-VP3 was constructed successfully, and the positive SGC-7901 cell clones expressing green fluorescent protein and fusion protein EGFP-VP3 stably were obtained. The fusion protein EGFP-VP3 has nucleic localization function in tumor cells and can induce the apoptosis of tumor cell.

关 键 词：凋亡素 融合蛋白 真核表达 胃癌细胞 

分 类 号：R730.23[医药卫生—肿瘤]