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

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.

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 SHG44. METHODS： By RT-PCR, the specific coding sequence fragments of the SVV 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-SVV 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, Western blot and immunocytochemistry. RESULTS： The SVV gene eukaryotic expression vector pcDNA3. 1-SVV was constructed successfully, which was identified by restriction endonuclease and DNA sequence analysis. Expression of SVV 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 SVV in SHG44 cells.