GFP-hTRAIL融合蛋白表达质粒的构建与卵巢癌细胞内表达  

Construction of GFP-hTRAIL fusion gene expression vector and its expression in ovarian cancer cells

作　　者：周莉[1] 丰有吉[1] 

机构地区：[1]复旦大学附属妇产科医院,上海200011

出　　处：《肿瘤》2005年第6期547-549,共3页Tumor

摘　　要：目的构建GFP-hTRAIL融合蛋白表达质粒,测定其在卵巢癌细胞中的表达。方法以GFP基因为报告基因,hTRAIL为目的基因,将hTRAIL基因插入GFP基因上游,构建成GFP-hTRAIL融合基因表达质粒,并通过酶切和测序对重组体进行鉴定。经脂质体法转染培养的卵巢癌细胞,用荧光显微镜观察GFP的表达。流式细胞仪分析转染后卵巢癌细胞的凋亡率。结果测序结果证实构建的为GFP-hTRAIL融合基因表达载体。质粒经脂质体转染后,有大约20％的卵巢癌细胞表达融合蛋白。流式细胞仪结果表明转染该质粒可诱导卵巢癌细胞凋亡。结论构建了1个可以表达GFP-hTRAIL融合蛋白的新质粒,该表达载体的构建成功为进一步开展卵巢癌的基因治疗奠定了基础。Objective To construct the fusion gene expression vector for EGFP and hTRAIL. Methods It was constructed for fusion gene expression vector, by inserting hTRAIL gene into N-terminus of GFP gene. The recombinant has been identified by the technology of restriction digest and electrophoresis. GFP-hTRAIL plasmid was transfected into ovarian cancer cells by LipofectamineTM 2000. Gene transfer efficiency was determined by fluorescent microscope and flow cytometry. Results Restriction analysis showed that the structure of the EGFP-hTRAIL plasmid was exactly the same as anticipated. Further resuhs indicated that both GFP and hTRAIL genes were simuhaneously expressed in ovarian cancer ceils. Apoptotic rate of ovarian cancer cell increased significantly by transfecting the plasmid. Conclusions A new plasmid has been established as the vector for studying the gene therapy of ovarian cancer. GFP gene expression can be used to monitor gene expression in living ceils. This study is the basic foundation for our next experiment.

关 键 词：卵巢肿瘤 肿瘤坏死因子 hTRAIL基因 遗传载体/生物合成 3AO细胞 

分 类 号：R737.31[医药卫生—肿瘤] Q782[医药卫生—临床医学]