摘要
目的构建包含人的G250真核表达载体,并将该载体转导入293 T细胞检测其表达,为构建以G250为靶点的肾癌模型提供研究基础。方法通过聚合酶链反应获得G250基因,将其连接至PMD18T过渡载体,然后克隆到载体pIRES-neo中。把构建后的重组质粒pIRES-neo-G250转染到293 T细胞,用流式细胞仪、免疫荧光和RT-PCR检测其表达。结果 PCR扩增出的G250基因测序正确,酶切鉴定重组质粒pIRES-neo-G250正确,说明质粒构建成功;流式细胞术、免疫荧光和RT-PCT检测结果表明,重组质粒pIRES-neo-G250在293 T细胞中得到表达。结论该实验成功构建了重组质粒pIRES-neo-G250,并且在293T细胞中能够有效表达,为后续构建转染人G250的基因细胞株工作奠定了基础。
Objective To construct the eukaryotic expression plasmid plRES-neo-G250 which expresses human G250, transduce it into the 293T cell and detect its expression. Methods The human G250 gene was amplified by PCR and linked into PMD18T, which was then inserted into the eukaryotic expression vector pIRES-neo. The constructed recombinant plasmid pIRES-neo-G250 was tansfected into the 293T cells, and its expression was determined by FACS, IMF and RT-PCR. Results The gene sequence of human G250 which was amplified by PCR was right. Enzyme digestion analysis re-vealed that the recombinant plasmid pIRES-neo-G250 was successfully constructed. FACS, IMF and RT-PCR revealed that the recombinant plasmid was expressed in 293T cells. Conclusion The successful construction of recombinant plasmid plRES-neo-G250 and its effective expression in 293T ceils can serve further construction of human G250-transfected cell line.
出处
《军事医学》
CAS
CSCD
北大核心
2013年第5期354-357,共4页
Military Medical Sciences
