重组真核表达载体pEGFP-BDNF的构建  

Construction of pEGFP-BDNF

作　　者：刘菲菲[1] 龙大宏[1] 龙静怡[2] 

机构地区：[1]广州医学院人体解剖学教研室,广东广州510182 [2]南方医科大学

出　　处：《解剖学研究》2012年第2期107-110,共4页Anatomy Research

基　　金：广东省自然科学基金项目(9151063101000016);广州市科技局应用基础研究计划项目(2009J1-C361-2)

摘　　要：目的将脑源性神经生长因子(BDNF)构建到表达质粒(pEGFP-N1)上。方法本实验采用RT-PCR的方法,从大鼠海马总RNA中扩增目的基因,分别用一步法(直接构建到表达载体上)和两步法(先构建到克隆载体再构建到表达载体上)构建重组载体,通过酶切和基因测序筛选鉴定正确的阳性克隆,并对两种方法做简单的比较。结果两种方法构建的重组载体都插入了正确的序列。两步法的转染效率明显高于一步法,但一步法也同样得到了正确的阳性克隆。结论成功构建了pEGFP-BDNF重组表达载体。Objective To construct pEGFP-BDNF.Methods BDNF gene was amplified by RT-PCR method from total RNA of rat hippocampus tissue,then the recombination vector system was constructed by one-step（constructed to expression vector directly） and two-step（construct to cloning vector firstly then to expression vector） approaches respectively.The right recombination was selected by enzyme digestion and identified by gene sequencing.Results The correct sequence was identified in both approaches.Transfection efficiency by two-step approach was much higher than one-step,but correct positive clones were also obtained by one-step.Conclusion Recombinant expression vector was constructed successfully.

关 键 词：脑源性神经生长因子 基因克隆 增强型绿色荧光蛋白 

分 类 号：R346[医药卫生—基础医学]