T7 RNA聚合酶表达系统的真核化及其偶联表达系统的建立

Eukaryonization of T7 RNA Polymerase Prokaryotic Expression System and Development of Its Couple Expression System

摘要:

为了使T7 RNA聚合酶（T7 RNAP）表达系统真核化，首先，利用两种方法建立能够表达T7 RNAP的真核细胞：（1）共转染表达T7 RNAP的真核表达重组质粒于靶细胞；（2）利用稳定表达T7 RNAP的BHK-21细胞系。然后，将FMDV内部核糖体进入位点（IRES）序列和增强型绿色荧光蛋白（EGFP）基因定向克隆进原核载体pET-40a-c(+)的T7启动子下游，得到的重组质粒pIERS-EGFP-E。用该质粒分别转染上述两种细胞，在紫外显微镜下都能够观察到绿色荧光，表明真核化的T7 RNAP偶联表达系统建立。并利用流式细胞仪对偶联表达水平进行了分析。这为利用该系统真核高效表达外源蛋白奠定了坚实的基础。用实验证明了FMDV 5′端含有IERS具有介导非帽依赖性的翻译的功能，为以IERS为基础的双顺反子表达系统的建立及深入研究IERS与相关蛋白的功能奠定了基础。T7 RNAP偶联表达体系是一种良好的外源基因表达体系。

Abstract:

To make transcription of the target gene be driven by T7 RNA polymerase (T7 RNAP) in the eukaryotic cells, and the transcripts be CAP-independent translated. Firstly, the T7 RNAP was introduced into eukaryotic cells by two methods: (1) the BHK-21 cells were contransfected by the plasmid expressing T7 RNAP and pIERS-EGFP-ET vector; (2) by transfection of the cell line stably expressing T7 RNAP. The internal ribosome entry site (IRES) element from FMDV was cloned into the downstream of the T7 promoter sequence of the prokaryotic expressing vector pET-40a-c(+), resulted in the plasmid would express the transcripts carrying the IERS element at its 5′ end. The enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IERS element, resulted in plasmid pIERS-EGFP-ET. Then, the two kinds of cells expressing T7 RANP were transfected by pIERS-EGFP-ET. The green fluorescence in the transfected cells was observed under a fluorescence microscope equipped with a video documentation system. And the expressional efficiency was analyzed with flow cytometry (FCM). The results show that the IRES element from FMDV has the role of initiating CAP-independent translation, and lay foundation for researching function of the element and interrelated proteins. It would be potential for expressing target gene by the T7 RNAP couple expression system.