摘要
目的 :克隆人Nanog基因 ,构建其真核表达载体 ,并观察其在哺乳动物细胞COS 7L中的表达。方法 :利用HE2 93细胞的人基因组DNA为模板 ,以LA PCR技术 ,扩增Nango的基因 ,定向克隆到带Flag标签的pCMV载体中 ,测序后 ,挑选序列正确的真核表达质粒pFlag Nanog转染COS 7L细胞。用抗Flag标签的抗体 ,进行Westernblot和间接免疫荧光染色法检测Nango蛋白的表达。结果 :从人基因组DNA中克隆到序列正确的Nanog全长编码序列。所构建的Nanog质粒在COS 7L细胞中获得高效表达。结论 :人Nanog基因的克隆、真核表达载体的构建及在COS 7L中的表达均获得成功 ,为进一步研究其功能 ,尤其是探讨其在神经干细胞中的作用奠定了基础。
AIM: To clone human Nanog gene and express it in mammalian cells. METHODS: LA-PCR was used to amplify human Nanog gene from genomic DNA of HEK 293 cells. The Nanog gene was inserted into the Flag-tagged pCMV vector by gene recombination technique. After being confirmed by sequencing, the pFlag-Nanog was transfected into COS-7L cells. Western blot and indirect immunofluorescent staining against the Flag tag were used to detect the expression of Nanog protein. RESULTS: The sequence of the cloned Nanog was confirmed to be correct by sequence analysis. The COS-7L cells transfected with pFlag-Nanog could efficiently express Nanog protein. CONCLUSION: The cloning of human Nanog gene and the construction of its eukaryotic expression vector were successful. The study will lay the foundation for further research on the function of Nanog and its role in neural stem cells.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第4期495-498,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金项目资助 (No .30 2 4 0 0 1 2 )
NSFC/RGC联合科研基金项目资助 (No .30 2 1 80 0 3)
关键词
人
NANOG
克隆
分子
真核表达
human
Nanog
cloning, molecular
eukaryotic expression
