Inhibitory effects of small hairpin RNA on hepatitis B virus X protein in vitro.

Objective To construct the shRNA expression vector targeting the hepatitis B virus X gene(HBX) and to observe the inhibitory effect of this vector on HBX protein expression in vitro.Methods A recombinant pSilencer3.1-shHBX plasmid,which can transcribe the shRNA targeting HBX,was constructed by cloning the annealed synthesized sequences into linearized pSilencer3.1-H1 vector and then named pSilencer3.1-shHBX.Then,pcDNA3.1-HBX and pSilencer3.1-shHBX were cotransfected into HepG2 cells by the liposome transferring method.Cells were cultured for 72 hours after transfection and RT-PCR and Western blot were performed to evaluate the inhibitory effect of pSilencer3.1-shHBX against the expression of the HBX gene and HBx protein,respectively.Results The recombinant pSilencer3.1-shHBX plasmid identified by restriction enzyme digestion and sequencing,was consistent with the design.As opposed to the negative control,the recombinant pSilencer3.1-shHBX plasmid efficiently inhibited the expression of HBX mRNA and HBx protein.The inhibitory rates were 47.1% and 58.9%,respectively.Conclusion The shRNA expression vector targeting HBX was successfully constructed and was able to inhibit the expression of the HBx protein.