pcDNA3.1-flag-pygo2真核表达载体的构建及在C6细胞中的表达  

Construction of eukaryotic vector pcDNA3.1-flag-pygo2 and expression in C6 cell

作　　者：王海东[1] 王占祥[1] 马永会[1] 谭国伟[1] 骆启聪[2] 程鹏[2] 

机构地区：[1]厦门市第一医院神经外科,福建厦门361001 [2]厦门大学生命科学院发育与肿瘤细胞实验室

出　　处：《中国实验诊断学》2008年第3期299-302,共4页Chinese Journal of Laboratory Diagnosis

摘　　要：目的构建pcDNA3.1-flag-pygo2真核表达载体并在C6细胞中进行表达。方法从小鼠脑胶质细胞中提取总RNA,RT-PCR法反转录合成c DNA,设计引物,调取目的片段,与pcDNA3.1-flag载体连接后转化大肠杆菌DH5α,LB平板筛选菌落,提取质粒。重组质粒pcDNA 3.1-flag-pygo2经过酶切鉴定及测序后,阳离子脂质体法转染C6细胞并经免疫细胞化学染色及蛋白印迹检测重组体的表达。结果重构质粒pcDNA 3.1-flag-pygo2经限制性核酸内切酶EcoRⅠ,HindⅢ酶切分析及测序检查,表明真核表达载体构建正确;瞬时转染C6细胞后,免疫细胞荧光染色及蛋白印迹检测表明转染细胞能够表达外源Pygo2基因。结论成功构建了pcDNA3.1-flag-pygo2真核表达载体并能够在真核细胞中进行表达,这为今后研究pygo2基因在胶质瘤中的作用机制奠定了基础。Objective To construct the eukaryotic expression vector pcDNA3.1-flag-pygo2 and express the combined protein in C6 cell line.Metgids To extract total RNA from primary glial cell of mouse and to synthesize c DNA by RT-PCR, then design primer and clone whole segment of pygo2 gene. After the targeted gene was inserted into vector pcDNA3.1-flag, the recombined plamid was transformed into E coli DH5α for LB agar plate screening and the recombined plasmid were extracted and purified. After verification by double enzyme digestion and sequencing., the constructed eukaryotic expression plamid was transfected to C6 cell line by lipofectamin method,finally the protein expression was detected by immunocytochemical staining as well as western blot. Results The new constructed vector was confirmed by restricted enzyme Eco R Ⅰ,Hind Ⅲ digestion assay and correct Pygo2 was verified by sequenceing, finally, pcDNA3.1-flag-hpygo2 can express exogenous pygo2 gene in glioma C6 cell line after transient transfection by the deterruination of immunocytotluorescent staining and western blot. Conclusion The new plamid pcDNA3.1-flag-pygo2 was constructed successfully, and can express fused protein in eukaryotic cell, which establish the foundation for future research on pygo2 gene function in human glioma.

关 键 词：pygo2基因C6细胞 载体 真核表达 WNT信号 

分 类 号：R346[医药卫生—基础医学]