【摘要】 目的:构建表达布氏田鼠催产素基因的慢病毒载体及过表达检测。方法:从布氏田鼠脑组织提取RNA逆转录PCR得到OT的cDNA片段,并分别连接到带有荧光素酶(luciferase,LUC)标记的慢病毒载体PGK启动子下游,以及带有绿色荧光蛋白(EGFP)标记的EF1A启动子下游,通过这两个载体转染细胞,分别利用生物发光标记与荧光标记这两种技术示踪OT基因的表达;再通过提取RNA逆转录以及实时荧光定量PCR检测OT基因不同模板转录的拷贝数。结果:成功构建LUC标记和EGFP标记的表达布氏田鼠催产素基因的慢病毒载体,进行了真核表达检测,并建立了实时荧光定量PCR的方法。结论:催产素(oxytocin,OT)基因是哺乳动物特有的神经垂体激素,与社会认知行为和社会适应行为有关,为研究该基因的功能奠定基础。更多还原

【Abstract】 Objective:To construct lentiviral vector of OT gene expression in Brandt’s vole and detect the expression quantity and expressed area of OT gene.Methods:Total RNA extracted from brain tissue of Brandt’s vole was amplified by RT-PCR for the cDNA fragments of OT gene and connected to two kinds of lentiviral vector,down stream of PGK or EF1A respectively.The two kinds of recombinant plasmid with OT gene labeling of luciferase,and eGFP marker respectivly,were transfected into 293T cells.The OT gene expression was detected in vivo by fluorescent imaging of luciferase or eGFP marker.At the same time,SYBR Green I real-time RT-PCR system was established to detect the copy number of OT gene in different samples.Results:The lentiviral vector labeled by LUC and EGFP was successfully constructed to express oxytocin gene of Brandt’s vole,and real-time fluorescent PCR method was also established to detect the copy numbers of OT gene in transgenic cells.Conclusion:Oxytocin gene is a peculiar neurohypophysial hormone of mammal,associated with social cognition behaviors and social adaption behaviors.更多还原