摘要
目的:构建MAD2基因与GFP融合的绿色荧光蛋白真核表达质粒,并在HepG2细胞中表达。方法:通过RT-PCR扩增,获得MAD2基因的完整序列。将此片断插入pEGFP-N1载体多克隆位点区,构建MAD2基因与GFP融合的绿色荧光蛋白真核表达质粒。应用脂质体转染技术转染HepG2细胞并观察表达的绿色荧光。结果:成功构建MAD2基因与GFP融合的绿色荧光蛋白真核表达质粒,并在HepG2细胞中观察到绿色荧光蛋白的表达。结论:该载体的成功构建,为研究MAD2基因在细胞分裂中的功能及定位建立了有效的观察体系。
Objective： To establish an in vitro screening system for testing effects of MAD2 gene of embryonic cell genome. Methods： In order to construct the plasmid expressing MAD2-GFP fusion plasmid, the target gene fragment was obtained by RT-PCR amplification and inserted into pEGFP-N1 reporter vector. The structure of constructed plasmid was confirmed by electrophoresis analysis and DNA sequencing. The function of construct was confirmed by lipofectamine-mediated transient expression in HepG2 cells. Results： DNA sequencing showed that the inserted fragment of the constructed plasmid was the same as the template MAD2 genome. The HepG2 cells transfected with the constructed plasmid could express reporter gene of gfp. Conclusions： the plasmid expressing gfp gene controlled by MAD2 gene is successfully constructed and an in vitro testing system for evaluating founction of MAD2 gene in separation of cell is established.
出处
《重庆医科大学学报》
CAS
CSCD
2006年第2期180-182,239,共4页
Journal of Chongqing Medical University
