布氏田鼠催产素基因的克隆及通过慢病毒载体转染细胞后的过表达检测  

Cloning of Brandt's vole oxytocin gene and its over-expression after cell transfection on lentiviral vector

作　　者：周文君[1] 施海霞[1,2] 宋铭晶[1,2] 

机构地区：[1]中国医学科学院医学实验动物研究所 北京协和医学院比较医学中心 卫生部人类疾病比较医学重点实验室,北京100021 [2]农业虫害鼠害综合治理研究国家重点实验室,北京100101

出　　处：《中国卫生检验杂志》2013年第8期1835-1838,共4页Chinese Journal of Health Laboratory Technology

基　　金：农业虫害鼠害综合治理研究国家重点实验室开放课题(ChineseIPM1204);北京市自然科学基金资助项目(7122110)

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。Objective： To construct lentiviral vector of OT gene expression in Brandt＇s vole and detect the expres- sion 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 fluores- cent imaging of luciferase or eGFP marker. At the same time, SYBR Green I real - time RT - PCR system was es- tablished 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. Condusion： Oxytocin gene is a peculiar neurohypophysial hormone of mammal, associated with social cognition behaviors and social adaption behaviors.

关 键 词：布氏田鼠 催产素 荧光素酶 绿色荧光蛋白 

分 类 号：Q754[生物学—分子生物学]