RhoCmiRNA真核表达载体的构建及其在卵巢癌细胞系中稳定表达.docVIP

RhoCmiRNA真核表达载体的构建及其在卵巢癌细胞系中稳定表达 作者：潘颖,毛丽君,孙艳霞,任淑萍,施恒亮,朱 单位：吉林大学第三临床医院妇产科，吉林 长春 【摘要】目的 体外合成RNA干涉小分子构建pcDNATM6.2GW/EmGFPRhoCmiRNA真核表达载体，分析其在人卵巢癌细胞SKOV3中稳定表达情况。方法 设计并合成单链oligo，经退火形成双链的DNA oligo，将其与线性的pcDNA6.2GW/EmGFPmiR质粒连接，构建重组质粒pcDNATM6.2GW/EmGFPRhoCmiRNA真核表达载体，经扩增、筛选、鉴定及测序后，用脂质体法将重组质粒转染SKOV3细胞，筛选稳定转染细胞系、观察转染细胞的形态并进一步通过Western印迹法检测细胞内RhoC蛋白表达。结果 重组质粒进行PCR鉴定及DNA序列测定证实DNA oligo已被成功连接到载体中并且序列完全正确；经重组质粒转染的SKOV3细胞24 h后的荧光显微镜下观察到少量细胞带有绿色荧光信号，RhoCmiRNA稳定转染的SKOV3细胞形态变圆，折光性增强，收缩不铺展。Western印迹结果显示，与SKOV3细胞株及pcDNATM6.2GW/EmGFPmiRneg细胞相比，pcDNATM6.2GW/EmGFPRhoCmiRNA细胞中RhoC蛋白表达水平明显降低，表明所设计的RhoCmiRNA能有效抑制SKOV3细胞中内RhoC蛋白表达。结论 该实验成功构建了RhoCmiRNA真核表达载体pcDNATM6.2GW/EmGFPRhoCmiRNA，利用脂质体法将其转染卵巢癌细胞后，能有效地抑制细胞内RhoC蛋白表达，为进一步研究RhoC的功能及探讨以RhoC为靶点的基因治疗提供了研究平台。 【关键词】 卵巢肿瘤；RhoC蛋白；小分子干扰RNA(RNAi)；真核表达载体 　　Construction of RhoCmiRNA eukaryotic expression vector and its stable expression in ovarian carcinoma cell 　　PAN Ying，MAO LiJun，SUN YanXia,et al. 　　Department of Gynecology and Obstetrics，ChinaJapan Union Hospital，Jilin University，Changchun 130033，Jilin，China 【Abstract】Objective To construct the eukaryotic expression vector pcDNATM6.2GW/EmGFPRhoCmiRNA and analyse its expression in ovarian carcinoma cell SKOV3.Methods Singlestrand oligo was synthesed and ligated with linear pcDNA6.2GW/EmGFPmiR to construct recombinant plasmid pcDNATM6.2GW/EmGFPRhoCmiRNA，after amplification，screening，determination and sequencing，it was transfected into SKOV3 cells.Screening was conducted to acquire the stable transfection strain and the expression of RhoC was confirmed by Westernblot.Results DNA oligo was successfully ligated with the expressing vector by DNA sequencing.SKOV3 cells were observed with green color after the transfected recombinant plasmid into the cells for 24 h under fluorescence microscope.The expression level of RhoC in SKOV3 cells transfected with pcDNATM6.2GW/EmGFPRhoCmiRNA was significantly decreased compared with that of SKOV3 cells and those transfected wi