人穿孔素基因的克隆及其在HepG-2细胞中的表达

• 作 者: 王立锋 许彦鸣 贾林涛 赵晶 温伟红 金明 王成济 杨安钢
• 机 构: 第四军医大学基础部生物化学与分子生物学教研室,第四军医大学基础部生物化学与分子生物学教研室,第四军医大学基础部生物化学与分子生物学教研室,第四军医大学基础部生物化学与分子生物学教研室,第四军医大学基础
• 出 处: 第四军医大学学报
• 发表日期: 2004年23期
• 关键词: 穿孔素 HepG-2细胞 表达

目的 :观察克隆的人穿孔素 (perforin ,PFN)基因在HepG 2细胞中的表达及其对转染的HepG 2细胞的作用 .方法 :用RT PCR方法获取人PFNcDNA .将所获基因克隆入真核表达载体pcDNA3.1-A ,转染HepG 2细胞 .间接免疫荧光法检测目的蛋白表达 ,电镜观察转染细胞的形态和结构变化 ,细胞计数检测转染目的基因后细胞的增殖情况 .结果 :成功获得人PFN全长cDNA ,并构建了真核表达载体 .转染HepG 2细胞后 ,检测出目的蛋白的表达 .细胞计数发现细胞增殖被明显抑制 .电镜观察显示 ,崩解的细胞呈现坏死的典型特征 .结论 :人PFN全长cDNA可以在转染的HepG 2细胞中表达 ,并且可使转染细胞的形态发生变化 ,出现大量细胞坏死 .

OBJECTIVE: To observe the expression of human perforin (PFN) gene in HepG 2 cells and its effect on transfected HepG 2 cells.Methods: The human PFN cDNA was obtained by RT-PCR.The cloned genes were cloned into real The recombinant plasmid pcDNA3.1-A was transfected into HepG2 cells.The expression of the target protein was detected by indirect immunofluorescence, the morphological and structural changes of the transfected cells were observed by electron microscopy, and the cell proliferation was detected by cell counting.Results: The full-length cDNA of human PFN was successfully obtained and the eukaryotic expression vector was constructed.The expression of the target protein was detected after transfected into HepG2 cells.The cell proliferation was significantly inhibited by electron microscope.The results of electron microscopy showed that the disintegrated cells were necrotic Typical characteristics.Conclusion: The full-length cDNA of human PFN can be expressed in transfected HepG2 cells, and the morphology of the transfected cells can be changed, resulting in a large number of cell necrosis.