稳定表达HBV C蛋白羧基端siRNA抑制HBV cccDNA的实验研究

目的研究乙型肝炎病毒(HBV)C蛋白羧基端短小的干扰RNA(siRNA)对HBV超螺旋双链闭环DNA(cccDNA)水平的影响.方法设计合成1对能稳定表达针对HBV C基因羧基端(2389nt-2410nt)序列的22-mer siRNA寡核苷酸,其两端带有限制性酶切位点,表达后可形成发夹结构,退火后形成双链;然后将这一双链寡核苷酸与重组腺相关病毒载体(rAAV)连接,构建重组体rAAV-siRNA.将重组体rAAV-siRNA加入培养增殖良好的22.1.5细胞中孵育,72 h后用实时聚合酶链反应(Real timePCR)法检测经处理过的22.1.5细胞和培养上清中HBV cccDNA的表达水平和HBV DNA的复制水平.结果重组体rAAV-siRNA实验组HBV cccDNA水平较空白对照组显著降低(P＜0.01),HBVDNA的复制水平在实验组亦明显下降;在无关序列对照组未发现对HBV cccDNA水平影响和HBV DNA复制的抑制作用.结论HBV羧基端siRNA能显著降低HBV cccDNA的水平和HBVDNA的复制.

Reduction of HBV cccDNA level by stably expressed HBV C protein C-terinal siRNA in vitro

Objective To study the effect of hepatitis B virus (HBV) C protein C-terminal small interference RNA (siRNA) on reduction of HBV covalently closed circular DNA (cccDNA) level. Methods A pair of oligos with sites of restriction endonuclease on their terminal was synthesized and annealed to form double-stranded oligo. Then the siRNA oligos were inserted into recombined adeno-associated viral vector (rAAV) for construction of recombinant (rAAV-siRNA), rAAV-siRNA could stably express short hairpin RNA which could be digested by dicer to become 22-mer siRNA complementary to the C-terminal site of HBV C protein (2389nt-2410nt). When 22.1.5 cells were treated with rAAV-siRNA 72 hours later, HBV cccDNA and HBV DNA levels in cultural supernatant and cells were determined by real-time PCR. Results The levels of HBV cccDNA and HBV DNA were significantly decreased in rAAV-siRNA group compared with blank control group (P< 0.01 ). No inhibitory effects on the levels of HBV cccDNA and HBV DNA in mock sequence group. Conclusion HBV C-terminal siRNA can decrease HBV cccDNA level and HBV replication in vitro.