人类pEGFP/H2B真核表达质粒的构建及表达

Construction and expression of the eukaryotic expression plasmid of Human pEGFP/H2B

目的:构建人H2B基因与GFP融合的绿色荧光蛋白真核表达质粒,并在HEK293细胞中表达。方法:通过RT-PCR从HEK293细胞总RNA中扩增获得H2B基因的部分cDNA序列,与载体pEGFP-C1连接构建H2B基因与GFP融合表达的真核表达质粒pEGFP/H2B,经酶切和测序鉴定;应用脂质体转染技术将重组质粒转染HEK293细胞观察融合蛋白的表达。结果:成功构建H2B基因与GFP融合的绿色荧光蛋白真核表达载体,与对照组中转染空载体pEGFP-C1的HEK293细胞中的弥漫荧光相比,转染重组质粒的细胞中可观察到绿色荧光蛋白集中表达在细胞核内,分裂期细胞中可见与染色体形态一致的绿色荧光。结论:该载体的成功构建为研究染色体的分离机制建立了有效的观察体系。

Objective：To construct the eukaryotic expression plasmid of EGFP/H2B and express it in the Human embryo kidney cells（HEK293）.Methods：Part H2B gene was obtained by RT-PCR from cultured HEK293 cell＇s whole RNA.The sequence encoding H2B was cloned into pEGFP-C1 to construct the eukaryotic expression vector.After confirmed by enzymatic digestion and sequencing,the recombination plasmid was transfected to HEK293 cells by lipofectamine technology for observation of the fusion protein＇s expression.Results：The recombination plasmid of pEGFP/H2B was constructed successfully.Compared with the dispersing green fluorescence among the whole cells in the control cells transfected with the mock plasmid（pEGFP-C1）,the green fluorescence was concentrated in the nuclei of HEK293 cells transfected with pEGFP/H2B and distributed with the chromosomes in the mitotic cells.Conclusions：The successful construction of pEGFP/H2B recombination plasmids establishes an available survey system to study the chromosome segregation mechanism.