人STIM1基因真核表达载体的构建及其在肝细胞中的表达  

Construction of human STIM1 eukaryotic expression vector and its expression in HL-7702 cells

作　　者：张振亚[1] 张宗明[1] 潘丽洁[1] 

机构地区：[1]清华大学医学院清华大学第一附属医院消化中心普外科,北京市100016

出　　处：《世界华人消化杂志》2009年第15期1489-1492,共4页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30270532;No.30670774;清华-裕元医学科学研究基金资助项目;No.20240000531;No.20240000547~~

摘　　要：目的:构建人STIM1基因真核表达载体,并观察其在人肝细胞株(HL-7702)中的表达.方法:提取HL-7702细胞总RNA,RT-PCR扩增,经纯化回收后将片段克隆至pGM-T载体,酶切琼脂糖凝胶电泳分析鉴定并测序,最后用重组质粒转染HL-7702细胞,通过PCR-电泳和Western blot法检测STIM1基因的表达.结果:PCR扩增的片段长度为342bp,测序结果以及重组质粒pGM-T-STIM1的酶切琼脂糖凝胶电泳鉴定结果均证明STIM1基因成功克隆到真核表达载体中.PCR-电泳和Western blot实验结果分别显示转染重组质粒后的HL-7702细胞在基因与蛋白水平表达STIM1均有所增强.结论:成功构建了人STIM1基因的真核表达载体pGM-T-STIM1,并在人肝细胞株HL-7702中稳定表达,为研究STIM1蛋白在人肝细胞内Ca2+浓度调控方面及其对人肝细胞分泌功能的影响奠定了良好的实验基础.AIM： To construct eukaryotic expression vector of human stromal interaction molecular 1 （STIM1） gene and to observe its expression in human liver cell line （HL-7702）. METHODS： Total RNA was extracted from HL-7702 cells and the coding sequence of STIM1 gene was amplified using RT-PCR. After purification, the fragment and eukaryotic expression vector pGM-T plasmid were ligated. The recombinant plasmid was verified by agarose gel electrophoresis and sequencing. Expression ofSTIM1 gene and growth of transfected HL-7702 cells were evaluated by electrophoresis and Western blot, respectively. RESULTS： The length of specific fragment amplified by PCR was 342 bp, the recombinant plasmid pGM-T-STIM1 showed two correct bands by digestion using restriction endonuclease EcoR, and sequencing results were as expected, suggesting that STIM1 gene had been cloned into pGM-T vector. Results of agarose gel electrophoresis and Western blot revealed that with the transfection, the expression level of STIM1 gene and protein were all elevated in HL-7702 cells. CONCLUSION： The recombinant eukaryotic expression vector pGM-T-STIM1 was successfully constructed and stably expressed in HL-7702 cells. It is meaningful for the research of cytoplasm calcium concentration regulation and secretion function in human liver cells.

关 键 词：STIM1 真核表达载体 人肝细胞株HL-7702 基因表达 

分 类 号：R575[医药卫生—消化系统]