摘要
目的:既往少数研究表明p120ctn存在核浆穿梭现象,为研究细胞核内p120ctn的功能,我们试图构建p120ctn 3A细胞核定向表达载体pCMV/p120ctn 3A。方法:采用PCR方法扩增人p120ctn 3A活性片段,将其插入细胞核定向表达载体pCMV/myc/nuc,构建重组体pCMV/p120ctn 3A,经脂质体介导转染肺癌细胞A549,用荧光显微镜观察绿色荧光信号,免疫细胞化学及Western blot鉴定其在肺癌细胞A549中的表达与定位。结果:限制性双酶切及DNA测序分析结果显示插入pCMV/p120ctn 3A的片段为2496bp左右,与预期p120ctn 3A基因片段大小相同。荧光显微镜观察到绿色荧光信号定位于细胞核内,免疫细胞化学结果显示表达的p120ctn 3A定位于A549细胞核。Western blot结果显示转染pCMV/p120ctn 3A的A549细胞p120ctn 3A蛋白表达量显著上调。结论:成功构建了p120ctn 3A细胞核定向表达载体pCMV/p120ctn 3A。
Objective：To construct p120ctn 3A nuclear target localizaiton plasmid.Methods： p120ctn 3A cDNA was amplified by PCR and inserted to nucleus-localization expression vector pCMV/myc/nuc to construct pCMV/p120ctn 3A.Then the pCMV/p120ctn 3A was transfected into A549 cells.The expression and localization of p120ctn 3A in A549 cells were verified by fluorescent microscope,immunocytochemistry and Western blot.Results： The size of inserted fragments in the recombinant vectors pCMV/p120ctn 3A was 2496bp,corresponding to that of p120ctn 3A.After transfecting pCMV/M-CSF into A549 cells by Lipofectamine 2000,pCMV/p120ctn 3A-transfected A549 cells could express p120ctn 3A protein in nucleus.The expression of p120ctn 3A was significantly elevated in pCMV/p120ctn 3A-transfected A549 cells.Conclusion： The nuclear localization vector pCMV/p120ctn 3A was successfully constructed.
出处
《现代肿瘤医学》
CAS
2012年第2期234-238,共5页
Journal of Modern Oncology
基金
国家自然科学基金资助项目(编号:30700806
81071717)
