摘要
目的:构建人CXCR4及CCR5真核表达重组质粒,转染人卵巢癌细胞SKOV3,建立稳定转染细胞系并观察其表达效果。方法:从人外周血单个核细胞中提取RNA,采用反转录PCR技术扩增CXCR4及CCR5的基因编码序列,将序列克隆至真核表达载体pEGFP,经酶切和测序鉴定后,应用脂质体转染技术将质粒cancer.pEGFP-CXCR4和pEGFP-CCR5分别导入不表达CXCR4及CCR5蛋白的SKOV3细胞,经G418抗性筛选得到阳性细胞克隆并扩大培养成系。分别采用免疫荧光染色和流式细胞术方法(FCM)检测稳定转染细胞株CXCR4和CCR5的表达。结果:构建了真核表达载体pEG-FP-CXCR4和pEGFP-CCR5;得到了抗G418阳性细胞克隆;免疫荧光染色和FCM检测结果显示,转染质粒的SKOV3细胞表达CXCR4和CCR5。结论:成功建立稳定表达趋化因子受体CXCR4和CCR5的卵巢癌细胞株,为CXCR4和CCR5在卵巢癌中的研究工作提供依据及平台。
AIM： To construct the eukaryotic expression vector of human CXCR4 and CCR5,transfect SKOV3 cells with them.METHODS： The cDNA of CXCR4 and CCR5 was amplified by RT-PCR.After purification,the gene was cloned into a vector pEGFP.The sequence of inserted CXCR4 and CCR5 gene fragment was identified by enzyme digestion of EcoR I/Sal I and sequencing,and then the recombinant plasmid was transfected into SKOV3 cells which did not express CXCR4 and CCR5 by lipofectamine-mediated gene transfection method.The SKOV3 cells transfected with CXCR4 and CCR5 were examined by FCM.RESULTS： Vectors pEGFP-CXCR4 and pEGFP-CCR5 were obtained.Cell clones which were screened by G418 were obtained.The results of FCM indicatedthat transfected SKOV3 cells expressed CXCR4 and CCR5.CONCLUSION： SKOV3 cells that can express CXCR4 and CCR5 protein stably have been established successfully,which facilitates the researches of epithelial ovarian.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2010年第12期1200-1202,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30872740)
