Construction and validation of a Gluc-GFP dual-labeled system

Abstract

Objective To construct a Gaussia luciferase-green fluorescent protein （ Gluc-GFP ） dual-labeled system for the investigation of in vitro and in vivo pathogenesis of virus infections .Methods Two DNA fragments encoding G1-10 and G11 were split from the gene encoding GFP and then respectively inserted into pcDNA3.1 vector to construct the recombinant expression plasmids G1-10-pcDNA3.1 and G11-pcDNA3.1.The genes encoding Gluc and G11 were linked by a fusion PCR and then inserted into pcDNA3.1 vector to construct the fusion plasmid.The expression plasmids of Gluc-G11-pcDNA3.1 and G1-10-pcDNA3.1 were transfected into 293T cells.The expression of Gluc in the supernatants was measured and a fluorescence microscopy was used to observe cells with GFP fluorescence.To simplify the construction process, a Madin-Darby canine kidney （ MDCK ） cell line consistently express G1-10 was constructed by Lentivector Expression Systems.The dual-labeled system was constructed by transfecting G11-pcDNA3.1 plasmid into the MDCK cell line.The puromycin and red fluorescent protein （ RFP） were used for screening and validation.Results The feasibility of split-GFP strategy was confirmed by using fluorescence micro-scope.The expression of luciferase in the supernatants and cells with green fluorescence could be detected after co-transfection of G1-10-pcDNA3.1 and Gluc-G11-pcDNA3.1 plasmids into 293T cells.The construc-ted MDCK cell line could stably express G1-10.Conclusion The physiological activity of Gluc was not af-fected by creating Glu-G11 fusion protein.The MDCK cell line stably expressing G1-10 could simplify the construction of the dual-labeled system.The successfully constructed Gluc-GFP dual-labeled system would provide a useful tool for further investigation on the pathogenesis of virus infections. Key words: Dual-labeled system Gluc-GFP Molecular imaging Virus research