一种新的双元表达质粒pCMV-Myc-IRES-EGFP的构建及其表达

Construction and Expression of a Novel Bisbicistronic Expression Vector: pCMV-Myc-IRES-EGFP

为了研究基因的特征、理化特性及其功能机制,通常需要构建多个真核表达载体,涉及到多次的引物设计、酶切、连接和鉴定等繁琐的亚克隆过程。构建携带易于多种实验研究的多用或通用载体是基因工程载体的发展方向。为此,利用pIRES载体为骨架质粒,在A和B多克隆位点上分别插入c-Myc标签蛋白序列和增强型绿色荧光蛋白(EGFP)序列,从而构建了一个包含c-Myc标签蛋白序列并携带有核糖体结合位点(IRES)介导的增强型绿色荧光蛋白的真核表达载体pCMV-Myc-IRES-EGFP。通过荧光检测和免疫印迹实验证实该载体能在哺乳细胞中表达。该载体可用于监测细胞的转染效率、分选稳定表达的阳性细胞群体、体外转录和翻译、检测或纯化目的蛋白以及捕获相关作用蛋白等多种实验研究,为基因功能研究提供了便利。

It is often necessary to construct more than one recombinant plasmids when investigating the characteristics,physchemical features and functional mechanisms of genes or proteins. Repeated sub-cloning procedures including design of primers, enzyme digestion,ligation and verification of recombinant plasmids, have to be involved with. For this reason, it has become a tendency to developing new genetic vectors which can be used in multitude of experiments. Therefore, by using plRES vector as a backbone, here we reported the construction of a mammalian expression vector： pCMV-Myc-IRES-EGFP which contains the Nterminal c-Myc epitope tag and the enhanced green fluorescent protein （EGFP） translated in an IRES-dependent manner. This novel vector can be used to testify the efficiency of cell transfection, to collect successfully transfected cell population via cytometry,to conduct transcription and translation in vitro, to purify target proteins or to trap their interactional proteins. The availability of this vector can facilitate function study of genes.