同济大学学报（医学版）2017，Vol.38Issue(1)：18-23,6.DOI:10.16118/j.1008-0392.2017.01.004
LOX-shRNA慢病毒表达载体的构建与鉴定
Construction and identification of LOX-shRNA lentiviral expression vector
摘要
Abstract
Objective To construct and identify the LOX-shRNA lentiviral expression vector.Methods.According to the sequence of human LOX gene,the shRNA sequence was designed and the sense and antisense oligonucleotides were synthesized.After annealing,these double DNA strands were inserted to the LOX expression vector and then transformed into competent cell E.coli TOP10.The plasmids were extracted and sequenced.The recombinant plasmid was transfected into HEK-293T cells and the expression level of LOX gene mRNA was detected by RT-PCR.After selecting the most effective LOX-shRNA,LipofectAMINETM2000 was used to transfect 293T cells for packing lentivirus and testing the titer of lentivirus.Results Recombinant plasmid LOX-shRNA-3024 was transfected into HEK-293T cells by lentivirus vector,and the recombinant plasmid was stably expressed.The green fluorescence HEK-293T cells was observed by fluorescence microscopy 24 h after transfection and reached the peak at 72 h after transfection,while there was no fluorescence was observed in untransfected HEK-293T cells.The titer of LOX-3024 recombinant lentivirus was 2 x 1010 TU/ml.Conclusion The LOX-shRNA lentiviral expression vector has been successfully constructed and stably expressed in HEK-293T cells,which can be used in studying the effects of LOX on the biological behavior of human vaginal wall fibroblasts.关键词
LOX基因/盆腔脏器脱垂/慢病毒载体/RNA干扰Key words
LOX gene/pelvic organ prolapse/lentiviral vector/RNA interference分类
医药卫生引用本文复制引用
周蒨,吴慧恒,陈信良,董晓燕..LOX-shRNA慢病毒表达载体的构建与鉴定[J].同济大学学报（医学版）,2017,38(1):18-23,6.基金项目
国家自然科学基金(81571419) （81571419）
上海交通大学医工交叉项目(YG2015MS40) （YG2015MS40）
