人穿孔素基因的克隆及其在HepG-2细胞中的表达

Expression of human perforin in HepG-2 cells

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 .

AIM: To observe the expression and the effect of human perforin (hPFN) protein on the HepG-2 cell line. METHODS: Human perforin full-length cDNA was obtained through RT-PCR from Jurkat cells, then it was cloned into vector pcDNA3.1 -A and the recombinant plasmid pcDNA 3.1 - A/hPFN was transfected into HepG-2 cells. The expression of hPFN gene was detected by immunofluorescent staining and the morphologic changes of HepG-2 cells subjected to the transfection of hPFN gene were detected by electronic microscopes. The proliferation of cells were found to be markedly inhibited after gene transfection. RESULTS: Human hPFN gene was cloned and inserted into pcDNA 3.1 - A vector. The hPFN protein was detected in the cytoplasm of transfected HepG-2 cells. Under electron microscope, the disintegrated cells displayed typical features of necrosis. CONCLUSION: Human perforin cDNA can be expressed in transfected HepG-2 cells and the expressed hPFN can lead to a morphologic change in cells characterized as necrosis.