广西医科大学学报2017，Vol.34Issue(5)：684-687,4.DOI:10.16190/j.cnki.45-1211/r.2017.05.011
CASK基因过表达慢病毒载体的构建及鉴定
Construction and identification of lentiviral vector over-expressing CASK gene
摘要
Abstract
Objective:To construct a lentiviral vector over-expressing calcium/calmodulin-dependent serine protein kinase (CASK).Methods:The whole length CASK gene fragment was amplified by polymerase chain reaction (PCR) and inserted into linearized GV358 lentiviral vectors using ligase.Positive clones of CASK-GV358 vectors were identified by PCR.The lentiviral vectors containing CASK gene were co-transfected into 293T cells with packaging plasmids.The virus titer was determined by ELISA.The recombinant lentivirus over-expressing CASK (LV-CASK) and negative control recombinant lentivirus CON238 were used to infect human NSCLC H1299 cells,respectively.Fluorescence quantitative PCR (FQ-PCR) and western blotting were used to detect the expression of CASK.Results:CASK gene was amplified and successfully inserted into the GV358 lentivirus vector.The sequences of the recombinant plasmid were confirmed by PCR and DNA sequencing.The expression of GFP could be detected in 293T cells infected with recombinant lentiviruses.The lentiviruses over-expressing CASK were harvested and concentrated to 2 × 108 TU/mL.According to the results of FQ-PCR and western blotting,the mRNA and protein expression level of CASK significantly upregulated in H1299 cell infected with LV-CASK,compared to cells infected with CON238 (P ＜0.05).Conclusion:The lentivriral vector over-expressing CASK gene was constructed successfully and stably expressed CASK in H1299 cells.关键词
CASK/慢病毒/载体构建/过表达Key words
CASK/lentivirus/vector construction/over-expression分类
医药卫生引用本文复制引用
覃觅,孙宇,刘俊,王延宁,赵文,周华富..CASK基因过表达慢病毒载体的构建及鉴定[J].广西医科大学学报,2017,34(5):684-687,4.基金项目
广西科技攻关计划基金资助项目(No.桂科攻14124004-1-4) （No.桂科攻14124004-1-4）
