稳定表达RAS鸟苷酸释放因子2肺癌细胞株的建立

Establishment of lung cancer cell strain for stable expression of RAS guanine nucleotide releasing factor 2

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 a eukaryotic expression vector for RAS guanine nucleotide releasing factor 2 （RASGR- F2）, tagged with GFP, and establish a lung cell strain for stable expression of RASGRF2. Methods Plasmid RASGRF2-pCMV6- Myc-DDK was digested with Sgf I and Not T , and RASGRF2 gene fragment was recovered and subcloned into vector pCMV6-GFP. Lung cancer H1299 cells were transfected with the constructed recombinant plasmid RASGRF2-pCMVS-GFP and observed for location of RASGRF2 protein under invert fluorescent microscope. The cell strain for stable expression of RASGRF2 was established by CAl 8 screening, and the expression of RASGRF2 was determined by RT-PCR and Western blot. Results Restriction analysis and sequencing proved that RASGRF2 gene was successfully cloned into eukaryotic expression vector pCMV6-GFP. Recombinant RASGRF2-GFP protein was mainly expressed in cytoplasm of H1299 cells. RT-PCR and Western blot showed that RASGRF2 was stably expressed in H1299 cells. Conclusion The lung cancer cell strain for stable expression of RASGRF2 gene was successfully established, which laid a foundation of further study on function of RASGRF2 gene.