靶向HBV和Hsc70基因的RNAi联合抗乙型肝炎病毒分子机制及其对天然免疫干预的研究

We report for the first time anti-HBV research that combines RNAi targeting HBV genome with that targeting heat shock cognate 70 gene. Our study found that siHBV+siHsc70 can highly effectively and specifically inhibit HBV replication and expression in HepG2.2.15 cells, and that inhibition of viral replication caused by siHsc70 targeting host protein gene does not depend on HBV genome itself. On the strength of previous research, we propose further study to illuminate the combinatorial anti-HBV molecular mechanism of siHBV+siHsc70. Analysis will be made of how siHBV and siHsc70 trigger innate immune type I IFN in transfected HEK293 and T98G cells; subsequently, upon cloning TLR3 genes from HEK293 and TLR7 genes from T98G, plasmid expression vectors for TLR3 and TLR7 will be transfected into HEK293 and T98G cells respectively. Expression vectors for siRNA will be transfected again and type I IFN expression will be appraised in order to determine the TLR receptor produced in the process of target gene-mediating siRNA triggering type I IFN. This will be an exploratory study of RNAi intervention with innate immunity, designed to provide a novel approach to developing prophylactic and treatment strategies against HBV.

我们首次报道将靶向HBV基因组和热休克同源蛋白（Hsc70）基因siRNA联合抗HBV研究，发现siHBV+siHsc70联用能够高效特异性抑制HBV在HepG2.2.15细胞中复制和表达,并观察到靶向宿主蛋白基因的siHsc70引起的病毒复制的抑制作用不依赖HBV基因组本身。在此工作基础上，本研究拟进一步研究试图阐明siHBV+siHsc70联用抗HBV分子机制；在转染的HEK293和T98G细胞中，分析siHBV和siHsc70诱发天然免疫I型IFN表达情况；克隆TLR3基因和TLR7基因，分别将TLR3和TLR7质粒表达载体转染到HEK293和T98G细胞内，再次转染siRNA表达载体并检测I型IFN表达，确认介导目的基因的siRNA诱发I型IFN产生的TLR受体。探讨RNAi联合对天然免疫干预，为研发HBV防治策略提供新思路。