RNA干扰对乙型肝炎病毒复制的体外影响

Inhibition of hepatitis B virus expression and replication by RNA interference

目的:通过一种能够在细胞内表达短发卡RNA的逆转录病毒载体来研究RNA干扰对乙型肝炎病毒复制的影响.方法:首先针对HBV基因组Pol基因RT区寻找RNAi的靶位,并设计相对应的寡核苷酸链.然后再将一对碱基配对寡核苷酸链退火后连接入载体形成重组的质粒,并进行鉴定.在Huh-7细胞中,将通过鉴定的干扰质粒与HBV复制型质粒pHBV3.8共转染,分别应用ELISA方法来检测HBV相关的抗原,应用Northern印迹检测HBV RNA,以及应用实时荧光定量PCR和Southern印迹检测HBV核心颗粒DNA.结果:研究通过计算机方法协助寻找了3条RNAi靶位,并且构建了相应的基于逆转录病毒载体的RNA干扰质粒154i、312i和734i.结果发现312i对pHBV3.8表达有明显的抑制,HBsAg和HBeAg分别为阴性对照组的39%和41%,差异均有显著性(P=0.001,P=0.000).定量荧光PCR结果显示312i组核心颗粒DNA水平显著低于阴性对照组(21.3%±1.1%vs 100.0%±10.6%,P=0.0046).Southern blot和Northern blot结果均显示,312i组病毒复制及mRNA转录水平最低(10.5%,12.0%).结论:在HBV基因组RT区找到了一个可用于RNAi的靶位,并且构建了相对应的干扰质粒,此质粒可成功地抑制HBV复制型质粒在细胞中的复制和表达.

AIM： To study the RNA interference on hepatitis B virus （HBV） replication by a reverse transcription virus vector which can express short hairpin RNA inside cells. METHOOS： pSIREN vectors with inserted oligonucleotides targeting on reverse transcriptase （RT） regions of HBV genome were constructed. These plasmids were co-transfected with pHBV3.8 into Huh-7 ceils. Viral antigens were measured by enzyme-linked immunosorbent assay （ELISA）. HBV core particle DNA was measured and quantified by real-time fluorescence quantitative polymerase chain reaction （RFQ- PCR） and Southern blot. Viral RNA was analyzed by Northern blot. RESULTS： Three RNA interfering targets were identified, and three corresponding retrovirus vectors, named 154i, 312i and 734i, were obtained. It was found that 312i markedly inhibited the expression of pHBV3.8, and the levels of HBsAg and HBeAg were 39% and 41% of those in the negative control group （P = 0.001, P = 0.000）. RFQ-PCR showed that the level of HBV core particle DNA was significantly lower in 312i group than that in the negative control group （21.3% ± 1.1% vs 100.0% ± 10.6%, P = 0.0046）. Southern and Northern blot demonstrated a lowest replication and transcription level of HBV in 312i group （10.5%, 12.0%）. CONCLUSION： A new RNAi system is identified in the RT regions of HBV genome, and the corresponding retrovirus vectors, which can remarkably inhibit the replication and expression of HBV, are also constructed.