摘要
目的:构建慢病毒转染的肥胖相关基因(fat mass and obesity associated gene,FTO)高表达体系,并建立稳定、高效、长久高表达FTO的人永生化膀胱移行上皮SV-HUC-1稳定细胞株,为研究FTO基因在膀胱癌发生及进展中的作用打下基础。方法:从SV-HUC-1细胞提取总RNA,逆转录合成cDNA。以cDNA为模板应用PCR技术扩增FTO编码基因,将其连接入PLEX-puro表达载体(PLEX-puro-FTO),并转化至感受态大肠杆菌JM109中。对氨苄青霉素和博来霉素双重筛选的阳性克隆进行测序,鉴定插入正确后抽提质粒,将阳性重组质粒导入病毒包装细胞293T中;收集病毒上清液转染SV-HUC-1细胞,48小时后加入终浓度1μg/ml的嘌呤霉素进行药物筛选,10天后得到稳定细胞株,用Western blot测定转染后SV-HUC-1细胞中FTO蛋白的表达水平。结果:测序结果证实目的片断正确插入PLEX表达载体中,成功构建了PLEX-puro-FTO高表达载体;稳定转染高表达载体SV-HUC-1细胞的FTO蛋白表达显著高于转染空载体细胞。结论:成功构建了PLEX-puroFTO重组慢病毒质粒,并获得FTO蛋白高表达的SV-HUC-1稳定细胞株。
Objective：To construct the expression system of lentiviral-mediated fat mass and obesity-associated gene（FTO）,and establish the human immortalized bladder epithelium SV-HUC-1cell with stable,efficient and long-term high expression of FTO in order to provide a basis for exploring the role of FTO gene in the development and progression of bladder cancer.Method：Total RNA was extracted from SV-HUC-1cells,and cDNA was synthesized by reverse transcription.FTO gene was amplified by PCR with cDNA as template.The PCR amplified FTO gene was ligated into PLEX-puro expression vector（PLEX-puro-FTO）,and transformed into competent E.coli JM109.The E.coli JM109 containing PLEX-puro-FTO vector were grown on LB agar plate supplemented with ampicilline and bleomycin by overnight.The single clone was picked for culturing overnight and then harvested for plasmid extraction.The recombinant plasmid was transfected into 293 Tcells,and the culture supernatant of 293 Tcells was collected,filtered and added to the SV-HUC-1cells.The cells were then selected by puromycin at a final concentration of 1μg/ml for over 10 days.The expression level of FTO in transfected SV-HUC-1cells were detected by Western blot.Result：Sequencing results showed that the target fragments were inserted correctly into PLEX expression vector,which means that we successfully constructed PLEX-puro-FTO vectors.Western blot results showed that the FTO protein expression levels in SV-HUC-1cells transfected with the recombinant plasmid were higher than the empty vector cells.Conclusion：The PLEX-puro-FTO lentiviral vector recombinant plasmid is successfully constructed,and the stable overexpression of FTO in the SV-HUC-1cells is successfully established.
出处
《临床泌尿外科杂志》
2015年第6期545-548,共4页
Journal of Clinical Urology
基金
国家自然科学基金(编号81272350
81472999)
广东省自然科学基金(编号S2012010008908)
广州市教育局基金(编号12A015G)
关键词
肥胖相关基因
膀胱癌
稳定细胞株
fat mass and obesity-associated gene
bladder cancer
stable cell lines
