【摘要】 目的:构建pEGFP-N1-Snail真核表达质粒,并进行鉴定,转染SCC25肿瘤上皮细胞,检测其表达。方法:利用RT-PCR方法从SCC9肿瘤上皮细胞中提取Snail基因片段,与pMD18-T载体连接,构建pMD18-T-Snail重组质粒,挑选阳性克隆,PCR鉴定后送测序。重组质粒双酶切后与pEGFP-N1真核表达载体连接,构建pEGFP-N1-Snail真核表达质粒,进行测序、酶切鉴定,表明质粒构建成功。分别抽提pEGFP-N1及pEGFP-N1-Snail质粒,用脂质体2000转染SCC25肿瘤上皮细胞,RT-PCR检测Snail在该细胞中表达。结果:本实验成功构建了pEGFP-N1-Snail真核表达质粒,并在SCC25肿瘤上皮细胞中表达。结论:本实验结果为进一步研究Snail作为转录抑制因子诱导上皮间质转化提供实验基础。更多还原

【Abstract】 Objective:To construct and identify a eukaryotic expression vector carrying Snail gene and test whether the plasmid could be expressed in the SCC25 cells.Methods:Snail gene fragment was amplified by using RT-PCR from SCC9 cells and was ligated into pMD18-T vector to construct pMD18-T-Snail. The transformed clones were amplified and sequenced. The resultant recombinant plasmid pEGFP-N1-Snail was confirmed by sequencing and using restriction enzyme. Then the pEGFP-N1 and pEGFP-N1-Snail plasmid were transfected into the SCC25 cells by Lipofectamine 2000 to test the expression of Snail by RT-PCR method. Results:The eukaryotic expression vector pEGFP-N1- Snail was constructed and expressed Snail in SCC25 cells. Conclusions:This provides a basis for further study on the function of Snail induces epithelial to mesenchymal transition as a transcriptional repressor.更多还原