A Minicircle DNA Vector-mediated SiRNA To Stably Suppress The Hbv Replication And Expression

siRNA (small interfering RNA) play a necessary factor in RNAinterfering (RNAi) effect,it is a21-25nt non-coding small RNA andprocessed by Dicer enzyme.siRNA is a main member ofRISC(RNA-induced silencing complex),which can inspire thecomplementary target mRNA silenced and participate inpost-transcriptional regulation of gene expression.siRNA has been shownto play an important role in many biological processes,but currently,themost difficult problem is how to transformed siRNA fragment into cells,inorder to expression target genes safely,efficiently and steadily.We utilized a minicircle DNA vector system to express siRNA andinvestigated the inhibition of hepatitis B virus (HBV) replication and geneexpression in vitro. siRNA targeting HBV S gene (siHBS) wasdesigned,synthesized and cloned into a minicircle DNA vectorpMC.BESPX-MCS2. After sequencing,we transformed the recombinantpMC-H1-siHBS-U6into E.coli ZYCY10P3S2T, and induced thedegradation of its bacterial backbone by adding L-arabinose into bacterial growth medium.As expected,a minicircle RNA interference vectorpmc-H1-siHBS-U6only consisting of gene expression cassette wasgenerated. Then pmc-H1-siHBS-U6was co-transfected into Huh-7cellswith HBV expression vector pHBV1.3. ELISA and Real-time PCR wereperformed to evaluate the inhibition effect of the secretion of HBsAg andHBeAg and the levels of HBV DNA and mRNA in Huh-7cells.We Successfully established the minicircLe-based RNAi vectorpmc-H1-siHBS-U6,which can significantly inhibit the secretion of HBsAgand HBeAg in Huh-7cells for two to three weeks. Real-time PCR resultsshowed that HBV DNA and mRNA levels were also down-regulated about71%and80%.The minicircle DNA-based RNAi vector pmc-H1-siHBS-U6can suppress HBV replication and gene expression specifically,efficientlyand steadily. Thus,this study provided us a new siRNA delivery system anda new gene therapy strategy of HBV infection.