摘要
采用PCR技术从小鼠cDNA文库中扩增出编码caveolin-1和flotillin-1基因片断,通过TOPO载体将基因克隆到表达载体pEGFP,转化至大肠杆菌DH5α中,抽提、纯化后将重组质粒转入PC12细胞中表达,用TIRFM观察高K+刺激下胞内标记蛋白的动态分布情况.实验结果显示重组质粒caveolin-1-pEGFP和flotil-lin-1-pEGFP在PC12细胞中具有一定的功能,而且caveolin-1和flotillin-1在发挥功能时可能有一个向膜转运的过程.
To detect the distribution of vesicle taking caveolin-1-pEGFP and flotillin-1-pEGFP, the DNA sequences of caveolin-1 and flotillin-1 from mouse cDNA bank were amplified with PCR technology. Utilizing TOPO middle vector, genes were cloned to the MSC of expression vector-pEGFP. After the two recombinant plasmids were transfected into PC12 cell, the intracellular distribution of themaeker proteins was observed on the 60 mmol K^＋ stimulation with TIRFM. The results showed recombinant plasmids of caveolin-1-pEGFP and flotillin-1-pEGFP had some function in PC12 cell. The dynamic process of the cell activity was observed firstly with TIRFM, which overcame the deficiency that the prevenient data were acquired from cells in static states. The experiments further showed that caveolin-1 and flotillin-1 might act to cell membrane, which have laid a foundation for the further study of the molecule function of caveolin-1 and flotillin-1.
出处
《华中科技大学学报（自然科学版）》
EI
CAS
CSCD
北大核心
2006年第1期118-121,共4页
Journal of Huazhong University of Science and Technology(Natural Science Edition)
基金
国家自然科学基金资助项目(30470646
30130230)
国家重点基础研究发展计划资助项目(2004CB720000)
湖北省自然科学基金资助项目(2003ABA096)
关键词
脂筏
小窝
PC12细胞
荧光标记
lipid raft
caveolae
PC12 cell
fluorescence labeling
