Construction of an expression vector mediated by the dual promoter for prokaryotic and mammalian cell expression system.

Abstract

The aim of this study was to construct an expression vector mediated by the dual promoter that can simultaneously drive the recombinant protein production in eukaryotic and prokaryotic cells. The prokaryotic T7 promoter and ribosome binding site (RBS) was cloned downstream of CMV promoter in the eukaryotic expression vector pIRES-neo, and T7 termination sequence was inserted upstream of neomycin phosphotransferase gene to generate the dual promoter vector. The enhanced green fluorescent protein (eGFP) gene was used as reporter gene. Then, the resultant vector was transfected into Chinese hamster ovary (CHO) cells and transformed into Escherichia coli (E. coli) BL21, and the eGFP expression levels were analyzed by fluorescence microscopy, flow cytometry and Western blot, respectively. Fluorescence microscopy revealed that the eGFP was expressed in both CHO cells and E. coli BL21. Flow cytometry showed that the eGFP expression level had no significant difference between the dual promoter vector and control vector in transfected CHO cells. Western blot analysis indicated the eGFP expressed in transformed E. coli. In conclusion, a prokaryotic-eukaryotic double expression vector was successfully constructed, which has potential applications in rapid cloning and expression of recombinant proteins in both prokaryotic and eukaryotic expression systems.