摘要
将c-flag-ago3质粒转入人293细胞系中,使c-flaga-go3基因稳定表达,为进一步研究AGO3蛋白复合物的结构、功能奠定了基础。利用亲和标签flag对目标蛋白进行检测和监测,将已合成的c-flag-ago3质粒和用于对照的质粒si-ago3(能使ago3基因沉默的质粒)导入人293细胞系中,在荧光镜下观察转染效果。RT-PCR法检测基因含量,蛋白印迹法(WB)检测人293细胞表达的flag-ago3,并对其蛋白复合物进行细胞定位。结果显示,c-flag-ago3质粒和si-ago3质粒成功地导入人293细胞系中,免疫细胞化学显示c-flag-ago3基因在细胞中高表达、并检测到AGO3复合物定位于细胞质中。AGO3复合物在细胞中可稳定表达,为进一步研究AGO3蛋白复合物的构成及其在人体中的功能奠定了基础。
It was to transfect c-flag-ago3 plasmid into human 293 cell line to make the c-flag-ago3 stable expression ,and investigate the structure and function of AGO3 protein compounds in human. The methods of affinity tags on the target protein were used ,then the c-flag-ago3 plasmid and the control si-ago3 plasmid which can cause ago3 gene silence were transfect into human 293 cell line, and the effect was observed through the fluorescence microscope, was observed RT-PCR detected gene content. Flag-ago3 protein was tested by western blot with mouse anti-flag Monoclonal antibody, and located the AGO3 protein compounds in cell. Results indicated that the c-flag-ago3 and the si-ago3 plasmid were inducted successfully in the human 293 cell line and the high expression of the ago3 was comfirmed by immuncytochemical method. It also proved that the AGO3 compounds located in the cytoplasm.
出处
《生物技术通报》
CAS
CSCD
北大核心
2009年第7期134-136,140,共4页
Biotechnology Bulletin
