Construction and identification of shRNA eukaryotic expression vector for HBx gene of hepatitis B virus

Objective To construct the shRNA eukaryotic expression vector for HBx gene of hepatitis B virus(HBV) and screen the interference sequence inhibiting HBx expression effectively.Methods Three siRNA expression vectors specific for HBx gene were designed and constructed,then identified by restriction analysis and sequencing.HepG2.2.15 cells were transfected with the constructed plasmid,then observed for transfection efficacy by fluorescent microscopy and determined for transcription level of HBx mRNA by RT-PCR and expression level of HBx protein by Western blot.Results The results of restriction analysis and sequencing showed that the plasmids were constructed correctly,and the sequence of inserted gene fragments were consistent with those designed.Both the transcription level of HBx mRNA and expression level of HBx protein in transfected HepG2.2.15 cells decreased significantly(each P 0.01),indicating that all the three plasmids inhibited the expression of HBx protein in HepG2.2.15 cells.However,the inhibiting ability of plasmid shRNA-HBx3 was higher than those of the other two plasmids.Conclusion The shRNA eukaryotic expression vectors for HBx gene were successfully constructed,of which the one effectively silencing HBx gene was screened.It laid a foundation of further study on onset and progress of HBx infection complicated with fatty degeneration in liver cells.