黏附调节因子p120ctn1A细胞核定向表达质粒的构建及鉴定

目的:构建并鉴定p120ctn 1A细胞核定向表达质粒pCMV/p120ctn 1A.方法:采用PCR的方法扩增人p120ctn 1A活性片段,将其插入细胞核定向表达载体pCMV/myc/nuc/GFP,构建重组质粒pCMV/p120ctn 1A,经脂质体介导转染肺癌细胞系SPC-A-1,用荧光显微镜观察绿色荧光信号,免疫细胞化学及蛋白质印迹法鉴定其在肺癌细胞SPC-A-1中的表达与定位,用Transwell小室法监测细胞侵袭能力的变化.结果:限制性双酶切及DNA测序分析结果显示,插入pCMV/p120ctn 1A的片段为2 798 bp左右,与预期p120ctn 1A基因片段大小相同.转染pCMV/p120ctn 1A质粒后,荧光显微镜观察到绿色荧光信号特异性定位于细胞核内.免疫细胞化学结果显示,SPC-A-1细胞核内p120ctn的表达量显著增强.蛋白质印迹法检测证实,转染pCMV/p120ctn 1A质粒后的p120ctn 1A蛋白表达量显著上调.体外侵袭实验结果证实,转染后SPC-A-1细胞的侵袭能力明显增强.结论:成功构建了p120ctn 1A细胞核定向表达载体pCMV/p120ctn 1A,为深入研究p120ctn的核内功能提供了有力的实验工具.

Construction and identification of p120ctn 1A nucleus-localization plasmid

OBJECTIVE: To construct p120ctn 1A nuclear target localizaiton plasmid.METHODS: p120ctn 1A cDNA was amplified by PCR and inserted to nucleus-localization expression vector pCMV/myc/nuc to construct pCMV/p120ctn 1A.Then the pCMV/p120ctn 1A was transfected into SPC cells.The expression and localization of p120ctn 1A in SPC-A-1 cells were verified by fluorescent microscope,immunocytochemistry and Western blot.Besides,the invasive ability of SPC-A-1 cells was detected by Transwell assay.RESULTS: The size of inserted fragments in the recombinant vectors pCMV/p120ctn 1A was 2 498 bp,corresponding to that of p120ctn 1A.After transfecting pCMV/M-CSF into SPC-A-1 cells by Lipofectamine 2000,pCMV/p120ctn 1A-transfected SPC-A-1 cells expressed p120ctn 1A protein in nucleus.The expression of p120ctn 1A was significantly elevated in pCMV/p120ctn 1A-transfected SPC-A-1 cells.Meanwhile,the invasive ability of SPC-A-1 cells was enhanced significantly.CONCLUSION: The nuclear localization vector pCMV/p120ctn 1A is successfully constructed.