Construction of GRbeta and GFP gene co-expressing lentivirus vector / 临床耳鼻咽喉头颈外科杂志

ABSTRACT

OBJECTIVE@#To construct the co-expressing lentivirus vector of human glucocorticoid receptor (GR) beta and green fluorescent protein (GFP).@*METHOD@#cDNA encoding hGRbeta obtained from the expression library of fetal brain using gene recombinant technology was cloned into pGC-LV by double digests technology. The constructed lentivirus vector of pGC-FU-GRbeta that was confirmed the sequencing was correct, transfected 293T cells through lipofectamine 2000. The constructed lentivirus vector was identified by fluorescence detection and Western Blot method. The packed lentivirus vector was used to infect 293T cells. The titer of virus was tested by real-time quantitative PCR.@*RESULT@#A recombinant lentivirus vector co-expressing hGRbeta and GFP gene was constructed successfully. After transfection, a large number of 293T cells with green fluorescence were observed under fluorescent microscope, and the expression of GRbeta and GFP fusion protein was detected by Western Blot. The virus titer was 2.00E+8 TU/ml tested by Real-time PCR.@*CONCLUSION@#Successful construction of hGRbeta and GFP co-expressing lentivirus vector provides a stable vector for investigating the relationship between GRbeta and hormonal sensitivity or resistance in the therapy of chronic rhinosinusitis whose choice drug are glucocorticoids.