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

作　　者：张峥嵘 郑幽[2] 阮班展 王嫚娜 梁剑青 李世崇[2] 黄红艳[3] 闫敏 陈昭烈 刘真真[1] 孙强 ZHANG Zheng-Rong;ZHENG You;RUAN Ban-Zhan;WANG Man-Na;LIANG Jian-Qing;LI Shi-Chong;HUANG Hong-Yan;YAN Min;CHEN Zhao-Lie;LIU Zhen-Zhen;SUN Qiang(Department of Breast, Cancer Hospital, Zhengzhou University, Zhengzhou 450008,China;Beijing Institute of Biotechnology, Beijing 100071,China;Beijing Key Laboratory of Oncology and Oncology Vaccine, Beijing Shijitan Hospital,Capital University of Medical Sciences, Beijing 100038,China)

机构地区：[1]郑州大学附属肿瘤医院乳腺科,河南郑州450008 [2]军事医学科学院生物工程研究所,北京100071 [3]首都医科大学附属北京世纪坛医院肿瘤内科肿瘤治疗性疫苗北京市重点实验室,北京100038

出　　处：《生物技术通讯》2017年第5期639-642,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81572799;31671432);国家重点研发计划(2016YFC1303303);北京市自然科学基金(7162091)

摘　　要：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.

关 键 词：Ras相关GTP结合蛋白D(RRAGD) 基因克隆 cell-in-cell 

分 类 号：Q78[生物学—分子生物学]