论文部分内容阅读
目的构建带有GFP标签的RAS鸟苷酸释放因子2(RAS guanine nucleotide releasing factor 2,RASGRF2)真核表达质粒,并建立稳定表达RASGRF2的肺癌细胞株。方法用SgfⅠ和NotⅠ双酶切RASGRF2-pCMV6-Myc-DDK质粒,回收RASGRF2基因片段,亚克隆入pCMV6-GFP载体中,构建重组表达质粒RASGRF2-pCMV6-GFP,转染肿瘤细胞H1299,倒置荧光显微镜下观察RASGRF2蛋白的定位。经G418筛选并建立稳定表达RASGRF2的细胞株,RT-PCR及Western blot法检测RASGRF2的表达。结果经双酶切及测序证实RASGRF2基因成功克隆至真核表达载体pCMV6-GFP中;重组RASGRF2-GFP蛋白在H1299细胞中主要在胞质中表达;经RT-PCR及Western blot证实,RASGRF2在H1299细胞中稳定表达。结论成功建立了稳定表达RASGRF2基因的肺癌细胞株,为进一步研究RASGRF2基因的功能奠定了基础。
Objective To construct eukaryotic expression plasmids of RAS guanine nucleotide releasing factor 2 (GAS) tagged with RASGRF2 and establish a lung cancer cell line stably expressing RASGRF2. Methods The RASGRF2-pCMV6-Myc-DDK plasmid was digested with SgfⅠ and NotⅠ, and the fragment of RASGRF2 gene was recovered and subcloned into pCMV6-GFP vector to construct recombinant plasmid RASGRF2-pCMV6-GFP. The recombinant plasmid was transfected into tumor cell H1299 and inverted fluorescence microscope The localization of RASGRF2 protein was observed. The cell lines stably expressing RASGRF2 were screened by G418 and the expression of RASGRF2 was detected by RT-PCR and Western blot. Results RASGRF2 gene was successfully cloned into eukaryotic expression vector pCMV6-GFP by double enzyme digestion and sequencing. Recombinant RASGRF2-GFP protein was mainly expressed in cytoplasm in H1299 cells. RT-PCR and Western blot confirmed that RASGRF2 was expressed in H1299 Stable expression in cells. Conclusion The lung cancer cell line stably expressing RASGRF2 gene was successfully established, which lays the foundation for further study on the function of RASGRF2 gene.