论文部分内容阅读
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 a high expression vector of lentivirus-transfected fat mass and obesity associated gene (FTO) and to establish a stable, highly efficient, long-term stable FTO-expressing human immortalized bladder transitional epithelial SV-HUC-1 stable cell line , To lay the foundation for studying the role of FTO gene in the occurrence and progression of bladder cancer. Methods: Total RNA was extracted from SV-HUC-1 cells and cDNA was reverse transcribed. The FTO-encoding gene was amplified by PCR using cDNA as a template, ligated into PLEX-puro-FTO and transformed into competent E. coli JM109. The positive clones screened by ampicillin and bleomycin were sequenced. The plasmids were identified after insertion and the positive recombinant plasmids were introduced into 293T virus packaging cells. SV-HUC-1 cells were collected and transfected into the SV-HUC-1 cells for 48 hours After adding puromycin at a final concentration of 1μg / ml for drug screening, stable cell lines were obtained after 10 days. The expression level of FTO protein in SV-HUC-1 cells was determined by Western blot. Results: The sequencing result confirmed that the target fragment was correctly inserted into PLEX expression vector and the PLEX-puro-FTO high expression vector was successfully constructed. The FTO protein expression in SV-HUC-1 cells transfected with high expression vector was significantly higher than that in transfected empty vector . Conclusion: The PLEX-puroFTO recombinant lentiviral plasmid was successfully constructed and the SV-HUC-1 stable cell line with high expression of FTO protein was obtained.