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

The aim of the study is to enable the transcription of the target gene be driven by T7 RNA polymerase (T7 RNAP) in the eukaryotic cells, and the transcripts be CAP-independent translated. First, 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 pIRES-EGFP-ET vector; (2) by transfection of the cell line stably expressing T7 RNAP. The internal ribosome entry site (IRES) element from foot-and-mouth disease virus (FMDV) was cloned into the downstream of the T7 promoter sequence of the prokaryotic expressing vector pET-40a-c(+), which resulted in the plasmid expressing the transcripts carrying the IRES element at its 5′ end. The enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IRES element, which resulted in plasmid pIRES-EGFP-ET. Then, the two kinds of cells expressing T7 RANP were transfected by pIRES-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 showed that the IRES element from FMDV has the role of initiating CAP-independent translation, and lays the foundation for researching the function of the element and interrelated proteins. It would be potential for expressing target gene by the T7 RNAP couple expression system.