凋亡抑制基因survivin真核表达载体的构建及其在SHG44细胞中的表达
Construction of survivin eukaryotic expression vector and its expression in SHG44 cells
张家墅 1甄海宁 1章翔 1张瑞 2韩涛 2左毅 1王鹏 1霍军丽1
作者信息
- 1. 第四军医大学西京医院全军神经外科研究所,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
- 折叠
摘要
目的:克隆细胞凋亡抑制蛋白基因survivin(SVV)的编码序列,构建含SVV基因的真核细胞表达载体并观察其在人胶质瘤细胞系SHG44中的表达.方法:利用逆转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)扩增SVV基因编码序列,扩增产物双酶切后定向克隆到真核细胞表达载体pcDNA3.1中,成功构建SVV基因真核表达载体pcDNA3.1-SVV.通过脂质体介导用pcDNA3.1-SVV转染SHG44细胞,经G418筛选,RT-PCR、免疫蛋白印迹(Western Blot)和免疫细胞化学方法鉴定SVV的表达.结果:经酶切鉴定和核苷酸序列分析,证实成功构建了SVV基因真核表达载体pcDNA3.1-SVV.蛋白和RNA水平检测表明SVV基因在SHG44细胞中稳定表达.结论:真核表达载体pcD-NA3.1-SVV使SVV基因在SHG44细胞中稳定表达.
Abstract
AIM: To clone coding sequence of human survivin (SVV) gene, to construct its eukaryotic expression vector and to observe its expression in human brain astrocytoma cell line SHC44. METHODS: By RT-PCR, the specific coding sequence fragments of the SW cDNA were amplified and cloned into eukaryotic expression vector pcDNA3.1. The eukaryotic expression vector pcDNA3.1-SVV was constructed and the gene transfection mediated by the lipofectin was used to introduce pcDNA3.1-SW into human brain astrocytoma cell line SHG44. The stable transfectant cells were established through selecting with G418. Expression of mRNA and protein of survivin was examined by RT-PCR, Westem blot and immunocytochemistry. RESULTS: The SW gene eukaryotic expression vector pcDNA3.1-SW was constructed successfully, which was identified by restriction endonuclease and DNA sequence analysis. Expression of SW gene in SHG44 was demonstrated by RT-PCR,Western blot and immunohistochemistry. CONCLUSION: Survivin gene has been successfully cloned and expressed in SHG44, which lays the experimental foundation for further study of biological functions and mechanisms of SW in SHG44 cells.
关键词
survivin/真核表达栽体/神经胶质瘤/SHG44细胞Key words
survivin/eukaryotic expression vectors/glioma/SHG44 cells引用本文复制引用
出版年
2009
NETL
NSTL
维普
万方数据