Fussenegger M, Mazur X, Bailey J E
Swiss Federal Institute of Technology, Institute of Biotechnology, ETH, Hönggerberg, HPT, CH-8093 Zurich.
Biotechnol Bioeng. 1998 Jan 5;57(1):1-10. doi: 10.1002/(sici)1097-0290(19980105)57:1<1::aid-bit1>3.0.co;2-m.
We constructed tricistronic expression vectors for the simultaneous and coordinated expression of three independent genes in mammalian cells. One single promoter allows high level and, in some vectors, adjustable transcription of all three cistrons. Whereas the first cistron is translated in a cap-dependent manner, the subsequent ones utilize intercistronic regions of viral origin such as the internal ribosomal entry site of poliovirus or the cap-independent translation enhancer of encephalomyocarditis virus for enhanced translation. Three multiple cloning sites with a total of up to 18 unique restriction sites allow sequential cloning of the genes of interest. The modular structure of this pBluescript(R)-based high copy number vector system allows straightforward movement of individual cistrons among members of the pTRIDENT family, and facilitates their combination with existing expression vectors.
我们构建了三顺反子表达载体，用于在哺乳动物细胞中同时且协调地表达三个独立基因。单个启动子可实现所有三个顺反子的高水平转录，在某些载体中还可进行调节。第一个顺反子以帽依赖方式进行翻译，后续顺反子则利用病毒来源的顺反子间区域，如脊髓灰质炎病毒的内部核糖体进入位点或脑心肌炎病毒的非帽依赖翻译增强子来增强翻译。三个多克隆位点共有多达18个独特的限制性酶切位点，允许依次克隆感兴趣的基因。这种基于pBluescript(R)的高拷贝数载体系统的模块化结构，使得各个顺反子能够在pTRIDENT家族成员之间直接移动，并便于它们与现有的表达载体相结合。
