Study on the characteristics and hepatotropism of the negative regulatory element of the HBVnt453-250 / 中华微生物学和免疫学杂志

ABSTRACT

Objective To analyze the characteristics and hepatotropism of this negative element HBVnt453-250 sequence.Methods pHBv453-250,pHBV250-453.plucHBV453-250 and plucHBV250-453 were constructed,with luciferase and enhanced green fluorecence protein(EGFP)gene as the reporter gene,respectively.After transfection of HepG2 cells with these plasmids,luciferase assays,real-time PCR and western blot assays were used to detect the gene transcription and expression level.The SV40 promoter of pGL3 control and pHBV453-250 were replaced by the cytomegalovirus early promoter,resulting in plasmids pCMVcontrol(luc)and pCMV453-250(luc).Results Compared with pHBV453-250,the mutant plasmids.with the inhibitory element inserted in difierent site or inverted orientation.exerted similar downregulation of Juciferase gene transcription and expression.Western blot analysis demonstrated the similar repression when EGFP was used as the reporter gene.By transfeeted to HepG2 cell line,the plasmid pCMV453-250(1UC)could reduce lneiferase activity(36.56%)compared with pCMLcontrol(luc).When the plasmids plueHBV453-250 and plucHBV250-453 were transfected to non-liver cell lines(A549,HeLa),luciferase gene was expressed weakly,compared with that of pGL3control(P＜0.05).Conclusion The inhibitory effect of HBVnt453-250 sequence acted in both orientation-and position-independent manners,and had no promoter selectivity and funotioned in hepatocyte-independent manner.