RRAGD-EGFP融合蛋白在MDA-MB-436细胞中的表达定位

Expression and Subcellular Localization of RRAGD-EGFP Fusion Protein in MDA-MB-436 Cells

目的:构建RRAGD-EGFP融合基因表达载体pQCXIP-RRAGD-EGFP,并检测RRAGD-EGFP融合蛋白在MDA-MB-436细胞中的表达定位。方法:提取HEK293细胞总RNA,逆转录得到cDNA并扩增RRAGD基因,克隆入逆转录载体pQCXIP-EGFP-N1,病毒包装后感染人乳腺癌细胞系MDA-MB-436,活细胞观察其在细胞内的表达定位。结果:构建获得逆转录病毒载体pQCXIP-RRAGD-EGFP,融合蛋白RRAGD-EGFP定位于胞浆囊泡和细胞核。结论:RRAGD-EGFP融合蛋白在MDA-MB-436细胞中表达定位于胞浆囊泡,与其参与溶酶体调节功能一致,为后续分析RRAGD在cell-in-cell中的作用奠定了基础。

Objective:To construct the retroviral expression vector for RRAGD-EGFP fusion protein,and examine its subcellular localization in MDA-MB-436cells.Methods:Total RNA was extracted from HEK293cellsand reverse transcribed into cDNA.RRAGD(Ras-related GTP binding protein D)gene was amplified by PCR andcloned into the retroviral expression vector pQCXIP-EGFP-N1to construct pQCXIP-RRAGD-EGFP,followed byretrovirus was made and infected MDA-MB-436,a cell line of breast cancer.Afterwards,time lapse imaging wasperformed.Results:The retroviral expression vector for RRAGD-EGFP fusion protein was successfully constructed.RRAGD-EGFP fusion protein was found in cytoplasmic vesicles and nucleus.Conclusion:Localization of RRAGDEGFPfusion protein in cytoplasmic vesicles is consistent with its role in lysosomal biogenesis,which has laid foundation for the further study on the role of RRAGD in cell-in-cell.