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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。
Objective: To construct a lentiviral vector expressing the oxytocin gene of Brandt’s viper and to detect its over-expression. Methods: cDNA of OT was obtained by RNA reverse transcription PCR from brain tissue of Brandt’s vole (Microtus brandti), and was ligated to the downstream of PGK promoter of luciferase (LUC) -labeled lentiviral vector and the green fluorescent protein EGFP) downstream of the EF1A promoter. The two vectors were used to transfect the cells, and the expression of OT gene was detected by bioluminescence and fluorescence labeling respectively. Then the RNA was reverse transcribed and detected by real-time fluorescence quantitative PCR Gene copy number of different template transcription. Results: The lentiviral vector expressing LUC and EGFP labeled Oxytocopus vomum gene was successfully constructed, and the eukaryotic expression was detected. The real-time PCR method was established. CONCLUSION: Oxytocin (OT) gene is a mammalian pituitary hormone that is related to social cognition and social adaptation behavior, which lays the foundation for studying the function of this gene.