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

作　　者：王立锋[1] 许彦鸣[1] 贾林涛[1] 赵晶[1] 温伟红[2] 金明[1] 王成济[1] 杨安钢[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033 [2]第四军医大学基础部免疫学教研室,陕西西安710033

出　　处：《第四军医大学学报》2004年第23期2113-2116,共4页Journal of the Fourth Military Medical University

基　　金：国家高科技研究与发展计划 (863)资助 (2 0 0 4AA2 1 70 70 ) ;国家自然科学基金 (30 2 0 0 2 74) ;国家杰出青年科学基金 (3992 50 36)

摘　　要：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.

关 键 词：穿孔素 HEPG-2细胞 表达 

分 类 号：Q255[生物学—细胞生物学]