作　　者： ; ; ; ;

机构地区： 教育部

出　　处： 《安徽农业科学》 2008年第35期15370-15372,15478,共4页

摘　　要： [目的]构建pEGFP-N1-hTERT真核表达载体,观察其在真核细胞中的表达。[方法]利用pC1-neo-hTERT和pEGFP-N1质粒构建重组质粒,通过双酶切鉴定、DNA测序分析验证人端粒酶逆转录酶基因(hTERT)片段的准确性。将pEGFP-N1-hTERT真核表达载体转染到大鼠胎儿神经干细胞(NSCs)中,通过绿色荧光蛋白间接观察人端粒酶逆转录酶蛋白在细胞中的表达定位,通过RT-PCR、WesternBlot验证所构建的真核表达质粒pEGFP-N1-hTERT的正确性。[结果]所构建的pEGFP-N1-hTERT真核表达载体结构正确并能够在真核细胞中表达。[结论]该研究为建立大鼠永生化NSCs系奠定了基础。 [ Objective ] The aim of this study is to construct eukaryotic expression vector pEGFP-NI-hTERT and observe its expression in eukaryotic cells. [ Method] The eukaryotic expression vector pEGFP-NI-hTERT was constructed with pCl-neo-hTERT and pEGFP-N1 plasmids, and the accuracy of human telomerase reverse trauscriptase （hTERT） gene fragment was confirmed by double enzyme digestion and DNA sequencing analysis. After transfecting the pEGFP-N1-hTERT eukaryotic expression vectors into rat fetal neural stem cells （NSCs）, the protein localization of human telomerase reverse transcriptase were indirectly observed through green fluorescent protein in the cells, and the correctness of constructed pEGFP-N1-hTERT was certificated by RT-PCR and Western Blot analysis. [ Result] The structure of pEGFP-N1-hTERT eukaryotic expression vector was correct and it could express in eukaryotic cells. [ Conclusion] This study laid a foundation for the establishment of immortalized NSCs line in rats.

关 键 词： 真核表达载体 构建 鉴定

领　　域： [农业科学]