摘要
通过PCR法扩增出N-Ras基因突变体片段,将酶切的片段克隆入真核表达载体pCDH-CMV-MCS-EF1-RFP中。经测序正确后转染293T细胞系,利用重组质粒PCR及串联基因表达的检测等方法对目的基因的转录与表达进行分析与鉴定。结果证实所构建的N-RasG12D基因突变体逆转录病毒载体目的基因序列完全正确,并能够在293T细胞中能瞬时表达。重组真核质粒的构建及表达为深入研究N-ras突变在癌症发病中的作用奠定了基础。
The target N-Ras gene mutation （G12D） were amplified by PCR and identified by enzyme digestion and then cloned into the eukaryotic expression vector pCDH-CMV-MCS-EF1-RFP. The recombinant plasmid was transfected into 293T cell line after sequencing. PCR was performed to determine the recombinant plasmid and then transient expresse of the gene was analysised by immunofluorescence to confirm the tandem gene were expressed. The recombinant plasmid is constructed successfully,and the gene is transient expressed in 293T cells. The stable transfected cell lines were constructed successfully, which laid a foundation for further study of the function of N-Ras gene mutation in earcer development.
出处
《科学技术与工程》
北大核心
2013年第13期3542-3544,3551,共4页
Science Technology and Engineering
基金
上海市科技发展基金项目(10140900800)资助
关键词
N-RAS
突变体
真核表达
N-Ras gene mutation eukaryotic expression
